Microbiologyhttp://hdl.handle.net/10125/2101
Fri, 09 Dec 2016 15:28:46 GMT2016-12-09T15:28:46ZUtilization of phosphatidylcholine, a lung surfactant component, as a major nurient source during Pseudomonas aeruginosa lung infectionhttp://hdl.handle.net/10125/100755
Abstract: Pseudomonas aeruginosa can grow to high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. Three different pathways, the betaine, glycerol and fatty acid degradation (Fad) pathways, are involved in the degradation of PC components including a phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs).
The Fad pathway still remains largely uncharacterized in P. aeruginosa. During the course of this work, fadBA1,4,5 operons (3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase) were shown to be the most important operons involved in fatty acid degradation through mutational analysis. Various fad mutants and the triple pathway mutant were analyzed extensively by in vitro growth analysis, virulence characterization, and competition study. Defect of growth on PC as sole carbon source was most significant on the triple pathway mutants, as expected. This growth defect translated to in vivo competition disadvantage in BALB/c mice, suggesting the importance of PC as nutrient source in vivo.
Description: M.S. University of Hawaii at Manoa 2013.; Includes bibliographical references.Sun, 01 Dec 2013 00:00:00 GMThttp://hdl.handle.net/10125/1007552013-12-01T00:00:00ZSun, ZhenxinNatural selection and the genetically encoded amino acid alphabethttp://hdl.handle.net/10125/101862
Abstract: Current science has advanced far beyond Crick's 'frozen accident' interpretation of the origin of the standard genetic code. Codon assignments can and do change, and new amino acids can be added to the code. Combined with the simple observation that the complex molecular machinery responsible for the standard code is a product of considerable evolution, it becomes legitimate and important to ask what else explains how and why one particular genetic code emerged within LUCA that still dominates the staggering diversity of life on our planet. Put another way, once we recognize the code as an evolvable phenomenon, we can ask what evolutionary forces shaped the emergence of the particular codon assignments found within the standard genetic code. Biological thinking has coalesced around three major ideas: the Adaptive Hypothesis, the Stereochemical Hypothesis, and the Biosynthetic or Co-Evolutionary Hypothesis.
Assessing the validity of all three theories (and any further estimation of their relative contributions) depends upon further investigations of two fundamental assumptions. These assumptions relate to the two previously mentioned chemical languages between which the genetic code acts as an interface: nucleotides and amino acids. A plethora of nucleotides and amino acids formed through biotic and abiotic processes were available in abundance during the earliest stages of life's evolution, as will be addressed in detail in Chapter 2. For the purpose of concluding this review of ideas regarding the evolution of the standard genetic code, what matters is to notice that any estimates made as to the relative importance of the theories described in this chapter build from the assumption of four nucleotides to encode twenty amino acids.
Description: M.S. University of Hawaii at Manoa 2013.; Includes bibliographical references.Wed, 01 May 2013 00:00:00 GMThttp://hdl.handle.net/10125/1018622013-05-01T00:00:00ZIlardo, Melissa AnnHetP and its three homologues : regions necessary for function of HetP and requirement of homologues for fixation of nitrogen in the filamentous cyanobacterium Anabaena sp. strain PCC 7120http://hdl.handle.net/10125/100599
Abstract: The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a Gram-negative prokaryote that performs oxygenic photosynthesis. In addition to being an obligate phototroph, Anabaena is capable of differentiating specialized nitrogen-fixing cells called heterocysts. The development of terminally-differentiated heterocyst cells occurs in the absence of fixed nitrogen and forms a one-dimensional pattern along the filament of vegetative cells. The exchange of intercellular signals controls the regulated spacing of the heterocyst cells that on average arise every tenth cell along the filament (Figure 1). The formation of heterocyst cells effectively separates the oxygen-labile nitrogenase complex from oxygen-evolving photosynthesis that occurs in vegetative cells. Heterocysts and vegetative cells are mutually interdependent. Heterocyst cells lack photosystem II and the capacity to fix carbon and must rely on the vegetative cells for sources of reductant. In return, heterocysts supply the filament with fixed nitrogen (Cumino et al. 2007; Marcozzi et al 2009). The development of two distinct cell types in a simple one-dimensional pattern makes Anabaena a simple example of cellular differentiation and pattern formation.
Description: M.S. University of Hawaii at Manoa 2013.; Includes bibliographical references.Thu, 01 Aug 2013 00:00:00 GMThttp://hdl.handle.net/10125/1005992013-08-01T00:00:00ZHurd, Kathryn LynnDetection of discordant isolates of drug resistant mycobacterium tuberculosishttp://hdl.handle.net/10125/101861
Abstract: Early diagnosis of mycobacterium tuberculosis is critical for proper treatment. With a regimen of rifampin and isoniazid, a patient with active tuberculosis can become non-infectious within 2 weeks (5). However, as resistance to these two drugs develops, second line antibiotic treatment must be used which lasts an extra 6 to 18 months and patients remain infectious for a longer period of time, which can further propagate the spread of mycobacterium tuberculosis (6).
Testing for antibiotic resistance in M. tuberculosis is a lengthy process due to its long generation time. The WHO standard guideline for the drug susceptibility test of M. tuberculosis is inoculating the bacteria through dilution on Löwenstein Jensen agar with a set concentration of test antibiotic and incubating it at 37 degrees Celsius. Colonies are then counted on the 28th day and a proportion is calculated by comparing the colony count of the test medium to a control. If the proportion exceeds a critical proportion or if no colonies appear in the lowest drug concentration medium with the highest inoculum, the isolate is determined to be resistant or sensitive, respectively. However, if neither criteria are matched, the incubation must continue until the 40th day where the final results are read through the same process (7).
The development of drug resistance strains in mycobacterium tuberculosis ultimately relies on exposure to the resistant drug because the presence of drug resistance mutations does not confer any selective advantage over strains lacking those mutations until exposure occurs (15). Within each geographical region, strains of M. tuberculosis may be under unique and various selective pressures to develop specific types of polymorphisms. Therefore, it can be hypothesized that there are genetic differences in the drug resistant strains of M. tuberculosis found in different regions and that the proportion of polymorphisms in genes associated with drug resistance will be different between geographic locations.
Study Objectives 1. To sequence the hot spot regions of nine genes (rpoB, inhA promoter, katG, ahpC promoter, gyrA, gyrB, rrs, eis promoter, and tlyA).
2. To identify discordant isolates through the comparison of drug susceptibility data and sequence data.
3. To further define the resistance patterns found in M. tuberculosis within and between different geographical regions represented by the widely dispersed study sites
Description: M.S. University of Hawaii at Manoa 2013.; Includes bibliographical references.Wed, 01 May 2013 00:00:00 GMThttp://hdl.handle.net/10125/1018612013-05-01T00:00:00ZHoshide, Matt KaneoViral vector construction, production and vector-mediated gene transductionhttp://hdl.handle.net/10125/102028
Abstract: Until now, viral vectors are considered necessary for gene therapy, and current approaches are prohibited from wide applications mainly due to low efficiency and genotoxicity. The use of optimized vector production systems, the right choice of target cells, and improved transduction protocols may overcome these obstacles.
To improve viral vector production, I initially optimized a calcium phosphate-mediated transfection method through inclusion of dextran and combined use of polybrene, and significantly improved the quality and quantity of the produce. Following that, multiple strategies, including a novel E. coli-based recombination system, Taq DNA polymerase treatment, and introduction of a bacteria toxic gene, were established and significantly improved the efficiency of generation of recombinant adenovirus vector. Moreover, multiple molecular manipulative strategies tested to a prototype retroviral vector system improved vector titers by 2-3 logs and led to enhanced transduction of a broad variety of cell types, especially cells of human and mouse haematopoietic and lymphocytic lineages that hold potential for gene therapy against a wide range of inherited and acquired diseases.
Furthermore, a series of mutant tRNALys3 genes were constructed and expressed using the optimized viral vector production systems, and showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication through improved priming of HIV-1 reverse transcription from their targeting sites. Transduction of multiple copies of mutant tRNALys3 further enhanced the anti-HIV-1 potency. Lastly, a soluble tumor necrosis factor-α receptor (sTNFR)-Fc fusion protein was designed and expressed to meliorate neurons through neutralizing TNF-α. TNF-α-binding activity of secreted sTNFR-Fc from transduced cells was demonstrated and conditioned medium containing sTNFR-Fc was shown to be protective to neuronal cells from TNF-α-, HIV-1 Tat-, and gp120-mediated neurotoxicity.
Overall, this study established multiple strategies and methods for improved viral vector production to facilitate gene therapy tests against HIV/AIDS and other diseases. The mutant tRNALys3-and sTNFR-Fc-based anti-HIV/NeuroAIDS strategies laid the groundwork for development of novel therapeutics against HIV and NeuroAIDS. Particularly, high efficiency transduction of cells of haematopoietic and lymphocytic lineages hold potential of using the genetically modified cells as noninvasive vehicles to deliver therapeutic substances across the blood-brain barrier into the central nervous system.
Description: Ph.D. University of Hawaii at Manoa 2013.; Includes bibliographical references.Wed, 01 May 2013 00:00:00 GMThttp://hdl.handle.net/10125/1020282013-05-01T00:00:00ZWu, ChengxiangUtilization of invasive algal biomass for bioethanol production and the dynamics of planktonic fungi in the West Pacifichttp://hdl.handle.net/10125/100650
Abstract: Algae represent the most promising feedstock for biomass derived biofuel production. Certain invasive algae in Hawaii can form dense biomass and are potential feedstocks for bioethanol production. In this study, the biomass from the invasive algae Gracilaria salicornia was used as feedstock for ethanol production using the ethanologenic strain Escherichia coli KO11. The algal hydrolysates were successfully utilized in a two-stage saccharification and fermentation platform, showing no inhibition of its bacterial fermenting ability, and producing 79.1 g ethanol from one kilogram of dry algal mass. Algae contain large quantities of species-dependent polysaccharides that cannot be readily metabolized by current ethanologenic bacteria. To fully explore the potential of microbial conversion of algal biomass and increase the systematic efficiency for ethanol production, culture-dependent and independent methods were applied to identify bacterial candidates fulfilling these purposes. The microbial communities profile associated with selected native and invasive algae were determined, which supplied valuable information in searching for candidates for polysaccharides utilization. Furthermore, microbes that can facilitate consolidated bioprocessing (CBP)--a process that can potentially optimize the systematic efficiency of biomass derived ethanol production--are isolated from various sources. Two bacteria FNP1 and TF2 showed great potential in further engineering for CBP platform development. Collectively, this study supplied valuable information in developing an efficient bioethanol production platform using invasive algal biomass.
The dynamics of planktonic fungi in the west Pacific was investigated in part II of the dissertation. This study revealed that planktonic fungi are molecularly diverse and the fungal distribution was related to major phytoplankton taxa and various nutrients including nitrate, nitrite, orthophosphate and silicic acid. Over 400 fungal phylotypes were recovered and nearly half of them grouped into two major novel lineages. Ascomycota and Basidiomycota were found to be dominant groups at majority of the investigated stations. These results suggest that planktonic fungi are an integral component of the marine microbial community and should be included in future marine microbial ecosystem models.
Description: Ph.D. University of Hawaii at Manoa 2013.; Includes bibliographical references.Thu, 01 Aug 2013 00:00:00 GMThttp://hdl.handle.net/10125/1006502013-08-01T00:00:00ZWang, XinMolecular epidemiology of seasonal and pandemic influenza A (H1N1) in Hawaiʻihttp://hdl.handle.net/10125/101981
Abstract: Influenza is a viral infection causing seasonal outbreaks, periodic epidemics and global pandemics in humans, the latest being the 2009 pandemic. The State of Hawaiʻi is particularly vulnerable to the spread of influenza due to its unique geographic position in the Pacific Ocean with heavily trafficked passenger and freight patterns. By combining epidemiological data on case occurrences with their laboratory-derived viral sequences, we are able to trace viral strain origins based on phylogenetic relationships between isolates.
In collaboration with the Hawaiʻi Department of Health State Laboratories Division, we present a study in which seasonal, or pandemic, H1N1 influenza A viral isolates collected from infected individuals in Hawaiʻi were extracted, hemagglutinin and neuraminidase genes were amplified and sequenced, and examined for evolutionary relationships and spatio-temporal patterns. Implications of molecular data are also supported by epidemiologic information and statistical support of summary transmission data. Phylogenetic analysis suggests that Hawaiʻi acts as both a source and sink population for type A influenza virus: in some instances Hawaiʻi isolates represented the earliest instance of a strain subsequently seen elsewhere; in other instances Hawaiʻi isolates clustered with strains observed earlier in other countries or geographic regions.
Through the continued usage of molecular methods, we hope to develop an improved understanding of influenza dynamics in Hawaiʻi. Targeting an area of geographic importance additionally assists in depicting how location and population distribution play a role in the spread of infectious disease. Enhanced comprehension as a result of these analyses may help to improve efficiency and effectiveness of preparation and response efforts, and reduce the impact of influenza on Hawaiʻi and the continental United States.
Description: Ph.D. University of Hawaii at Manoa 2013.; Includes bibliographical references.Wed, 01 May 2013 00:00:00 GMThttp://hdl.handle.net/10125/1019812013-05-01T00:00:00ZNelson, Denise CynthiaThe epidemiology and entomological interactions associated with dengue transmission in Ang Mo Kio GRC, Central Singaporehttp://hdl.handle.net/10125/100707
Abstract: Dengue is arguably the most important arboviral disease of humans, having increased dramatically in geographic range and prevalence over the last 25 years. Dengue virus has two main vectors, Aedes aegypti and Aedes albopictus. For decades both vectors have also been increasing their geographic range on regional and global scales. This study took place in Singapore, where dengue fever is a major public health threat despite a successful vector control program. Similar to other hyperendemic countries, local dengue transmission dynamics in Singapore are not well understood: where dengue transmission is occurring, the relative contribution of the two dengue vectors, and the ability to correlate traditional vector surveillance methods to transmission risk remains controversial. In collaboration with the Program of Infectious Diseases at Duke-NUS Graduate Medical School, Singapore Ministry of Health, Singapore National Environmental Agency, and Ang Mo Kio Town Council an adult Aedes female fixed position vector surveillance program was established that detailed temporal and spatial Ae. aegypti and Ae. albopictus distribution and abundance in Ang Mo Kio, Central Singapore. This surveillance method yielded similar results to standard surveillance techniques over a range of habitats and time points. Furthermore, sensitivity of the adult surveillance method presented here is uniquely increased by placing traps on the second floor of Housing Development Board (HDB), government subsidized multistory residential buildings, as opposed to ground level; average Ae. aegypti catch rate of the ground floor was 0.09 and average Ae. aegypti catch rate of the second floor was 0.42. Starting on the second floor a very strong inverse relationship between Ae. aegypti catch rate and floor height (Pearson linear correlation r=-0.91, t=-4.47, df=4, p=0.01) was also identified. In addition, intensive entomological investigations, in focal areas with varying levels of Aedes abundance, identified by the fixed position surveillance system, uncovered details about mosquito ecology and "hotspots" at a local scale that can improve our understanding of dengue transmission dynamics. Dengue transmission is believed to primarily occur in residential units but host seeking Ae. aegypti and Ae. albopcitus were collected at similar frequencies in congregation areas on the ground floors of, HDBs and at greater abundance than inside residential units. Improving knowledge on the focal nature of dengue transmission is critical to designing more targeted and cost-effective surveillance and control strategies in the future, both in Singapore and urban areas elsewhere.
Description: Ph.D. University of Hawaii at Manoa 2013.; Includes bibliographical references.Sun, 01 Dec 2013 00:00:00 GMThttp://hdl.handle.net/10125/1007072013-12-01T00:00:00ZHenry, Amy BethDevelopment of a non-enzyme based capillary dipstick assay for the detection and purification of environmental pathogenshttp://hdl.handle.net/10125/101671
Abstract: Novel mAbs were produced with a wide range of anti-Dickeya binding reactivities. A mAb line produced from the first fusion generated a BOX-PCR type B,C and D binding antibody (MAb Pine-1). A second mAb line had broad spectrum specificity that reacts to BOX-PCR type A through E and most of the Dickeya reference strains (MAb Pine-2). Phylogenetic analysis based on gyr-B gene expression and BOX-PCR types are consistent with the mAb reactivities.
Also a field dipstick assay was developed for the detection of live environmental pathogens that is able concentrate the organism for further analysis and was able to detect S. enterica serotype Typhimurium at 105 CFU/ml, which is within the infective dose of most Salmonella species. The dipstick is able to isolate and purify S. enterica serotype Typhimurium from a mixed culture of 100:1 S. marcescens to S. enterica serotype Typhimurium, with a final purity of greater than 80% determined by colony counts, which is culturable to streak for identification.
Description: M.S. University of Hawaii at Manoa 2010.; Includes bibliographical references.Wed, 01 Dec 2010 00:00:00 GMThttp://hdl.handle.net/10125/1016712010-12-01T00:00:00ZLuu, Van PhiNumerous fatty acyl-CoA synthetase homologues are involved in fatty acid degradation in pseudomonas aeruginosahttp://hdl.handle.net/10125/101720
Abstract: The goal of this research is to characterize the newly identified fatty acyl-CoA synthetase homologue, PA3860 (fadD3), which is thought to take part in Fad though its exact contribution to fatty acid metabolism in P. aeruginosa is unknown. Deciphering the role of fadD3 in fatty acid degradation will broaden our knowledge of the β-oxidation pathway in this bacterium. This was achieved by completing the following specific aims: 1. Purification of FadD3 and performance of biochemical analysis of its acyl-CoA synthetase function 2. Genetical characterization of fadD3 (PA3860) of P. aeruginosa through mutational analysis and gene fusion studies During the course of this research additional discoveries about the β-oxidation pathway of P. aeruginosa were made: 1) additional acyl-CoA synthetase homologues were found, i.e. fadD4, fadD5, and fadD6 2) and their role in Fad was determined along with fadD3.
Description: M.S. University of Hawaii at Manoa 2011.; Includes bibliographical references.Sun, 01 May 2011 00:00:00 GMThttp://hdl.handle.net/10125/1017202011-05-01T00:00:00ZZarzycki-Siek, Jan BozydarThe prevalence and public health significance of human pathogenic vibrio species (v. cholerae, v. vulnificus, v. parahaemolyticus, v. alginolyticus) in Hawaiʻi's diverse tropical coastal water environmentshttp://hdl.handle.net/10125/101779
Abstract: Studies on the prevalence and ecology of Vibrio species in tropical areas, such as Hawaii, is limited, and up to now, there have been no studies conducted in Hawaii to determine the prevalence of these pathogens in our coastal waters. The major goals of this study was to determine the prevalence of the four human pathogenic Vibrio spp. (V. cholerae, V. vulnificus, V. parahaemolyticus, V. alginolyticus) in coastal water environments of Hawaii (islands of Oahu and Hawaii), and to determine the public health significance these pathogens have to people who use these coastal water for recreational purposes.
The study showed that water salinity and temperature affected the four human pathogenic Vibrio spp. V. vulnificus and V. parahaemolyticus were prevalent in low salinity sites that were impacted by land run-off but not detectable in high salinity, non-impacted swimming sites. Both species were also prevalent at low salinity swimming ponds on the island of Hawaii. V. alginolyticus was prevalent in all sites regardless of salinity.
In addition to low salinity, high water temperature also had an impact. High temperature, low salinity ponds located on the Island of Hawaii were shown to select for V. vulnificus, V. parahaemolyticus and V. alginolyticus. These ponds have shown past evidence of infection and death due to V. vulnificus associated with the use of these ponds. Isolates recovered from these thermal ponds may potentially be more virulent as they have been adapted to survival at temperatures similar to that of human body temperature. V. cholerae was not recovered in either impacted or non-impacted sites.
The prevalence of pathogenic Vibrio spp. in sediments followed a similar trend to what was seen with coastal beach samples. V. alginolyticus was prevalent in both primary and secondary beach sediment while V. vulnificus and V. parahaemolyticus were only prevalent in secondary beach sediment. Thus, apparently sediments from secondary coastal waters can spread pathogenic Vibrio species into the water column.
Data from this study also showed that V. vulnificus and V. parahaemolyticus were sporadically present in raw and primary treated sewage from three different wastewater treatment plants, while V. cholerae was consistently recovered in raw and primary treated sewage from all three treatment plants. V. vulnificus can cause severe wound infections, which can rapidly lead to death. Thus, this species poses a public health significance.
In summary, data gathered from this study was able to provide basic information, that was lacking, regarding the distribution of the four main human Vibrio pathogens in a tropical area such as Hawaii. This data was then used to make a basic assessment of the potential public health significance these pathogens have on humans who use Hawaii's coastal waters for recreational purposes, and to determine if and when warning signs would be warranted to notify the public of the potential risk for infection.
Description: Ph.D. University of Hawaii at Manoa 2011.; Includes bibliographical references.Sun, 01 May 2011 00:00:00 GMThttp://hdl.handle.net/10125/1017792011-05-01T00:00:00ZVithanage, GayatriBioprospecting for thermostable glycoside hydrolases : a metatranscriptomic approachhttp://hdl.handle.net/10125/100858
Abstract: The conversion of cellulosic material from linear form to monomer is the rate-limiting step in current biofuels production technologies from lignocellulosic material. The renewed focus on clean, sustainable, carbon-neutral fuels has resulted in increased interest in novel cellulolytic enzymes and microbial strains, with the aim to increase the efficiency of the above conversion. However, the availability of suitable cellulolytic enzymes has been restricted by the limited number of cellulolytic microbial strains in which these enzymes can be procured. One approach to increase efficiency has been to bioprospect novel thermophilic microbial strains, the logic being that an increase in production temperature will result in increased rates of lignocellulosic hydrolysis. Unfortunately, microbiologists have had limited success in cultivating cellulolytic thermophiles with only a small number of strains isolated.
The goal of this project was to circumvent the need to cultivate cellulolytic thermophiles by applying a metatranscriptomic approach to discover new thermostable cellulases commonly referred to as glycoside hydrolases (GH-enzymes that hydrolyze the glycosidic linkages between sugar molecules). This was achieved by employing a novel in situ enrichment technique to enrich for thermostable GHs directly from a geothermal environment. These GHs were being actively expressed by the resident microbial population to hydrolyse lignocellulose. This approach not only eliminated the need for cultivation, but also selected for the actively expressed GHs unregulated in response to the lignocellulosic material feedstock. This strategy removed the emphasis on identifying potentially relevant and substrate-active GHs from genomic data via genomic analysis of a cultivated microorganism or from environmental metagenomic surveys.
Description: M.S. University of Hawaii at Manoa 2012.; Includes bibliographical references.Sat, 01 Dec 2012 00:00:00 GMThttp://hdl.handle.net/10125/1008582012-12-01T00:00:00ZLyford, Jeffrey RossIn silico and in vitro characterization of ten putative RsbR-like proteins in Saprospira grandishttp://hdl.handle.net/10125/101306
Abstract: Globin-coupled sensors (GCSs) are heme-binding proteins that consist of an N-terminal sensor globin domain and a C-terminal transducer domain. Their functions are varied depending on the C-terminal domain and can be classified into two groups such as aerotactic and gene regulation groups. The gene regulating group is further divided into DNA-binding, 2nd messenger and protein-protein interaction group. So far, aerotactic transducers: HemAT-Hs from Halobacterium salinarum and HemAT-Bs from Bacillus subtilis are well characterized in a molecular level and a cellular level. In addition, GCSs from the 2nd messenger group within the gene regulating group such as BpeGreg (Bordetella pertussis), EcGreg (E. coli), CvGreg (Chromobacterium violaceum) and AvGreg (Azotobacter vinelandii) are recently characterized as well.
Recently, genome sequencing of Saporospira grandis has been completed and revealed in-depth genomic information that related to interesting traits of this organism such as gliding motility, production of rhapidosome and ixotrophic nutrient obtaining mechanisms. Moreover, the initial machine annotation showed that there are ten putative GCSs which contain C-terminal STAS domains. Based on the machine annotation results, we have ten genes that were predicted to be GCSs and have a C-terminal STAS (Sulfur transporter and anti-sigma antagonist) domain (Table 1.1). The putative function of these proteins is an anti-sigma factor antagonist like SpoIIAA, RsbR and RsbS in B. subtilis which involves in nutritional, physical and environmental stress responses. The protein named RsbR1 (SGRA_3210) within S. grandis was first discovered and its neighbor genes rsbS,-T,-V,-X and rsbU shared similarities with rsbR and its adjacent genes (rsbS,-T,-U,-V,-W, sigB, rsbX) in B. subtilis. Also, the structure of RsbR consists of the N-terminal non-heme globin domain and the C-terminal STAS domain. All these genes and their encoded proteins are related to activate more than 150 stress response genes upon physical stresses through releasing a sigma factor B. Based on these similarities, we postulate RsbR1 and nine other proteins are globin-coupled sensors that are involved in the stress response.
In this study, in silico and in vitro characterization of ten GCSs were carried out on both the N-terminal globin and the C-terminal STAS domains. Multiple alignments of the globin domains showed that only SGRA_0571 (RsbR2), SGRA_3210 (RsbR1) and SGRA_3852 (RsbR3) aligned their histidine residue with the proximal histidine residue in known globin domains. Two phosphorylation sites from RsbR and its paralogs in B. subtilis were aligned with nine GCSs except SGRA_3852 and YtvA which is known not to have any phosphorylation site, instead it has a GTP binding motif. Two amino acid residues (D and G) from the GTP binding motif (DXXG) are all aligned in all GCSs. The 3D structure alignments of the globin domain of Geobactor sulfurreducens and RsbR1, RsbR2 and RsbR3 showed around 23% identity but the proximal histidine, where it held the heme, were all aligned with the histidine residue of three GCSs. For in vitro characterization, all ten genes were cloned, expressed and purified. The purified proteins were dialyzed and carried UV spectroscopic analysis. Only three GCSs (RsbR1, RsbR2 and RsbR3) exhibited oxygenated myoglobin like peaks. This shows that these proteins are able to bind oxygen. RsbR1 was undergone for crystallization. Initial crystallization screening showed some red crystals.
Initially we found ten GCS genes from the annotation of the genome. In silico results predicted them all as GCSs. Multiple alignments of the globin domains and STAS domains of ten GCSs showed only RsbR1, RsbR2 and RsbR3 had the conserved proximal histidine residues which were vital for heme-binding. The STAS domains of ten GCSs were shared high homology with the STAS domains of RsbR and its paralogs from B. subtilis including two conserved phosphorylation sites and possible GTP-binding motifs. However, RsbR3 did not show the conserved phophrylation sites. All ten GCS genes were cloned, expressed and purified. RsbR1, RsbR2 and RsbR3 displayed oxygenated myoglobin-like absorption spectra and they were the only proteins that were validated as GCSs.
Description: M.S. University of Hawaii at Manoa 2012.; Includes bibliographical references.Tue, 01 May 2012 00:00:00 GMThttp://hdl.handle.net/10125/1013062012-05-01T00:00:00ZKim, Sun AeModulation of TNF-α production by small RNA in dengue virus infected human monocytic cellshttp://hdl.handle.net/10125/100961
Abstract: The immunopathology of dengue virus (DENV) infection is associated with increased TNF-α production. In this study, small RNA-mediated regulation of TNF-α and the effect of TNF-α knockdown during DENV infection were analyzed. This provides insight into the role of TNF-α during DENV infection, both in terms of its contribution to immunopathogenesis and its regulatory mechanism by miRNAs.
Utilizing a lentiviral expression system, human monocytic U937 cells that express short hairpin RNAs designed to target TNF-α mRNA were established. TNF-α expression was downregulated in these monocytes, and upon DENV infection they showed decreased endothelial cell activation ability. This demonstrates an overall decrease in the proinflammatory response upon TNF-α knockdown during DENV infection.
To analyze the role of microRNAs (miRNAs) in the TNF-α response, miRNAs that potentially target the 3' UTR of TNF-α were predicted. Many of the miRNAs were differentially regulated during DENV infection. miR-320a and miR-592 were among those downregulated, and chosen for further analysis. TNF-α post-transcriptional regulation by miR-320a and miR-592 was confirmed utilizing a TNF-α 3'UTR luciferase reporter. U937 cells transfected with miR-320a and miR-592 mimics followed by DENV infection displayed decreased TNF-α expression and their culture supernatants demonstrated decreased ability to activate endothelial cells. It is concluded that one function of these miRNAs is to negatively regulate TNF-α, and their downregulation contributes to the immunopathogenesis of DENV infection.
Description: M.S. University of Hawaii at Manoa 2012.; Includes bibliographical references.Wed, 01 Aug 2012 00:00:00 GMThttp://hdl.handle.net/10125/1009612012-08-01T00:00:00ZHammond, Kimberly ElaineEffective concentration and detection of human enteric viruses in Hawaiian environmental watershttp://hdl.handle.net/10125/101292
Abstract: Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the aquatic environment.
Reported here are novel and effective laboratory protocols for enhanced enteric virus detection in Hawaiian environmental waters. First, a fine-tuned, highly optimized assay for the detection of enterovirus, an important enteric virus subset, was developed by comparatively evaluating eighteen published enterovirus primer pairs for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated through testing urban wastewater, and then utilized in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites tested positive for enterovirus, indicating fecal contamination in a significant portion of Hawaiian waters.
Additionally, the filter-feeding phenomenon of indigenous bivalve mollusks was explored as a natural bioconcentration technique to infer microbial quality of the surrounding waters. Shellfish were collected from 12 coastal locations and dissected for subsequent nucleic acid extraction from internal tissues. Optimized RT-PCR/PCR protocols were then applied to test for the presence of various enteric viruses, including enterovirus, adenovirus, norovirus genogroups I and II, and F-specific RNA coliphage. Shellfish collected from around the island tested positive for several enteric virus types, indicating that these animals are indeed natural and competent bioindicators of water quality. The extremely sensitive and innovative techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii's environmental waters.
Description: M.S. University of Hawaii at Manoa 2012.; Includes bibliographical references.Tue, 01 May 2012 00:00:00 GMThttp://hdl.handle.net/10125/1012922012-05-01T00:00:00ZConnell, Christina NoelleCharacterization of biocontaminants in biodiesel fuels and potential roles in the formation of microbially induced corrosionhttp://hdl.handle.net/10125/100908
Abstract: Biological contamination of renewable biodiesel fuels is a recognized problem that requires detailed investigations (Passman, 2001). Increased demand for energy independence and viability as a fossil fuel alternative has rapidly expanded use of biodiesel globally. However, there are several problems associated with biodiesel. One such problem is biological contamination, which reduces fuel stability and induces corrosion.
At the start of this project, it was hypothesized that metal corrosion and fuel degradation by microorganisms are cometabolically enhanced by biodiesel in blended fuels. To investigate this hypothesis, experiments were designed to characterize microbial contaminants in biodiesel fuels and study their potential roles in microbial degradation, microbially induced corrosion and cometabolism.
Description: M.S. University of Hawaii at Manoa 2012.; Includes bibliographical references.Sat, 01 Dec 2012 00:00:00 GMThttp://hdl.handle.net/10125/1009082012-12-01T00:00:00ZChing, Travers Han-mingPolyphasic characterization of an epilithic biofilm from a lava cave in Kīlauea Caldera, Hawaiʻihttp://hdl.handle.net/10125/100817
Abstract: The microbial community in an epilithic biofilm on an lava cave wall in Kīlauea Caldera, Hawaiʻi, was characterized by a polyphasic approach. Ribosomal-pyrotag and metagenomic sequencing revealed phylogenetic diversity rivaling that in a Guerrero Negro hypersaline microbial mat. Targeted cultivations led to the isolation, characterization, and genome sequencing of a deeply divergent novel cyanobacterium. Diverse Bacteria and Archaea lineages were detected. The most abundant sequences, representing ~24% of the metagenomic reads analyzed, affiliated with Burkholderia. Comparative metagenomic analyses revealed community composition and function most similar to those in soils. Two novel cyanobacteria detected in metagenomic data were cultivated; JS1 is related to Gloeobacter violaceus PCC 7421T , the only cultivated Gloeobacter species. JS2 may represent a new genus in the Oscillatoriales since it shares <95% 16S rRNA gene sequence identity with its nearest neighbor, a Leptolyngbya sp. A third cultivated cyanobacterium (JS3) not detected in clone libraries, ribosomal-pyrotag or metagenomic data sets, belongs in the true-branching filamentous Stigonematales; JS3 shares 98.1% 16S rRNA gene sequence identity with Fischerella muscicola PCC 7414, and may be a new Fischerella sp.
Comparing the complete genome sequence of JS1 with that of G. violaceus PCC 7421T revealed JS1 represents a new species, despite sharing 98.7% 16S rRNA gene sequence identity with PCC7421T . The name Candidatus Gloeobacter kilaueaensis is proposed, with JS1T the Type strain. Maximum likelihood phylogenetic trees based on 16S rRNA gene sequences and 43 concatenated ribosomal proteins showed Candidatus Gloeobacter kilaueaensis JS1T places in the deep-branching Gloeobacter clade, but is less basal than G. violaceus. Divergence times based on Bayesian analyses suggested these Gloeobacter species diverged 150-300 MYA. The isolation, characterization, and genome sequencing of a deeply divergent novel Gloeobacter is significant given that for forty years we have known only one species in the entire order. Of broader significance is confirmation that by integrating molecular and cultivation methods we can target for cultivation specific Bacteria and or Archaea only detected in molecular analyses; a range of scripts was also developed to analyze and visualize sequence data.
Description: Ph.D. University of Hawaii at Manoa 2012.; Includes bibliographical references.Sat, 01 Dec 2012 00:00:00 GMThttp://hdl.handle.net/10125/1008172012-12-01T00:00:00ZSaw, Jimmy Hser WahThe ecology of planktonic bacteria in oligotrophic marine systemshttp://hdl.handle.net/10125/101357
Abstract: A broad synthesis regarding bacterioplankton ecology in the oligotrophic ocean was obtained through investigations along three fundamental ecological units--the community, the population and the organism. Community investigations focused on the spatial and temporal dynamics of the bacterioplankton at Station ALOHA, a site representative of the oligotrophic North Pacific subtropical gyre. Microbial DNA was harvested from water samples collected at monthly intervals over a 4-year period from surface to 4000 m depths. A suite of 16S ribosomal RNA gene-based techniques including massively parallel pyrosequencing, terminal restriction fragment length polymorphism and clone library sequencing were used to interrogate the bacterioplankton community. Distinct communities were observed stratified within the water column, with a major distinction near the base of the euphotic zone and finer distinctions through water mass association. These communities appeared structured through K-selection, as they were dominated by a surprisingly few number of specific taxa. Weak seasonality was detected in surface communities that resulted from fluctuations in relative abundances of rare populations. These rare populations were associated with the phytoplankton community and correlated with mild seasonal environmental disturbances. Low diversity was observed in both surface and bottom waters and highest in mesopelagic waters near the oxygen minimum zone. These observations fit an intermediate disturbance model that is based upon high frequency environmental disturbances at the surface and lower frequency disturbances at depth in the form sinking particulate matter, as taxonomic evidence supports. Taken together, the oligotrophic bacterioplankton community is viewed to consist of bulk members that are highly competitive for limited resources and rare opportunists that exploit habitat patchiness. The overall stability of these communities in time and space is dependent upon the integration of autochthonous (water mass) community with the allochthonous (particle associated) community. Major populations representative of the autochthonous community were investigated in further detail, yielding results that suggest their abundances may be controlled by basic physical parameters, such as temperature. Investigations at the organismal level focused on an isolate representative of the allochthonous community at T=0, as its abundances are correlated with diatom blooms. Genomic interrogation uncovered physiological features that enhance our view of oligotrophic bacterioplankton ecology.
Description: Ph.D. University of Hawaii at Manoa 2012.; Includes bibliographical references.Tue, 01 May 2012 00:00:00 GMThttp://hdl.handle.net/10125/1013572012-05-01T00:00:00ZHayakawa, Darin HideoMetagenome recruitment of the global ocean survey dataset to four closely-related SAR11 genomeshttp://hdl.handle.net/10125/100300
Abstract: The free-living marine bacterial clade known as SAR11 is thought to be one of the most abundant lineages of organisms on our planet. It is also one of the smallest free-living organisms, and possibly the ancestor of the very successful endosymbiont, mitochondria. Members of this group of bacteria possess an incredibly small genome that displays a high degree of synteny and high DNA recombination rate, which represents the upper limit for bacteria. This study examines the global metagenomic read recruitment of the genomes of four closely related SAR11 strains, HIMB4, HIMB5, HIMB83, and HIMB140, in publicly available coastal and open ocean metagenome libraries. Recruitment plots were used to identify and analyze clusters of genes with unusual recruitment patterns, and utilized 2,797,042 open ocean and 2,131,159 coastal high quality reads. It was found that strain HIMB83 possessed the most highly recruited genome and had the highest coastal preference of the four. A hypervariable region previously identified in SAR11, HVR2, was identified in all four strains, and was characterized by a large number of genes coding for cell wall and membrane components as well as several enzymes involved in carbohydrate metabolism. HVR1 was also present in all examined genomes where it was dominated by 12 proteins involved in the type II secretion and type IV pilus pathway. The recruitment of the proteorhodopsin gene present in each of the strains was similar, with a greater degree of conservation on the 3' end of the gene. A phage integrase and extra DNA polymerase was found within the genome of HIMB4, suggesting a possible lysogenic virus present within the genome. This study adds to the current understanding of SAR11 subgroup 1a in a global context, and provides a workflow for generating metagenome recruitment plots for future studies.
Description: M.S. University of Hawaii at Manoa 2014.; Includes bibliographical references.Thu, 01 May 2014 00:00:00 GMThttp://hdl.handle.net/10125/1003002014-05-01T00:00:00ZBrucks, Eric SnodgrassLipid body function as non-classical calcium stores in immunocyteshttp://hdl.handle.net/10125/101219
Abstract: Lipid bodies, found in most eukaryotic cells, are intracellular lipid storage organelles. The role of lipid bodies has been most studied in adipocytes and hepatocytes due to their role in energy storage. Comparatively, little is known of the role they play in immunocytes. Both in adipocytes/hepatocytes and in cells of the immune system, lipid body numbers are dynamically regulated and can accumulate to pathophysiological levels (steatosis). In both locales this steatotic state can be induced by nutrient overload and metabolic stress, and in the latter also under certain conditions of infection. The over-arching goals of this project are (1) to study the composition (lipid and protein) and functional contributions made by lipid bodies in immunocytes, and (2) to assess the impact of altered lipid body numbers upon cellular function. The work presented in this thesis describes three areas of progress towards these goals. First, we developed a microaspiration method for isolating highly purified lipid bodies, allowing for their ex vitro manipulation and study of lipid/protein content using microscopy. This technique was validated using fluorescence microscopy of the neutral lipid dye Oil Red O in microaspirated lipid bodies. Second, we assessed the impact of the accumulation of lipid bodies (steatosis) on transcytoplasmic calcium signaling, a major activation pathway in the model immune cell system studied here. Third, we tested a new hypothesis arising from our work on transcytoplasmic calcium signaling. The apparent ability of lipid bodies to act as long term loci for calcium accumulation, coupled with recent studies showing that lipid bodies may contain mitochondria and endoplasmic reticulum, led us to hypothesize their potential role as bona fide calcium stores. Our data revealed that the lipid body population within mast cells is not homogenous. However, some LB exhibit the ability to sequester calcium and to release it in the manner of a bona fide calcium store. These observations represent a potentially novel role for lipid bodies and indicate the possibility of their contribution to calcium dynamics in immune cells under both physiological and pathophysiological conditions.
Description: M.S. University of Hawaii at Manoa 2014.; Includes bibliographical references.Mon, 01 Dec 2014 00:00:00 GMThttp://hdl.handle.net/10125/1012192014-12-01T00:00:00ZPhan, Nolwenn KathrynCellular response of insect cells to virus infectionhttp://hdl.handle.net/10125/100551
Abstract: RNA interference (RNAi) is the dsRNA-triggered gene regulatory mechanism that is evolutionally conserved in most eukaryotic cells. It has been widely used as a powerful tool for functional genomics in various organisms. In flies, mosquitoes or other insect cells, gene functional analysis by RNAi is usually performed through introduced dsRNAs that are synthesized by in vitro transcription.
RNAi serves as an important innate immunity against viruses in plants and invertebrates. It has recently been shown that Aedes albopictus mosquito C6/36 cells, commonly used for arbovirus propagation, possess an impaired RNAi pathway. In this study, we developed in vitro Dicer assay using extracts prepared from mosquito cells. Our results confirmed the inability of C6/36 cells to process dsRNAs into siRNAs, which is consistent with the loss-of-function of Dcr-2 due to a frameshift mutation. However, such a defect could not be complemented by introduction of Drosophila Dicer-2. To evaluate the RNAi-based antiviral mechanism in C6/36 cells, we analyzed the replication of a mutant Nodamura virus (NoV) genomic RNA1 of which viral RNAi suppressor B2 is not expressed (NoVR1ΔB2) and cannot accumulate to a detectable level in RNAi-competent cells. In C6/36 cells, the defective RNAi gives rise to complete restoration of NoVR1ΔB2 replication, suggesting that RNAi is the primary antiviral immunity in mosquito cells.
At present, dsRNA, as the trigger of the antiviral RNAi pathway in invertebrate and plants, is the major efficiency limitation factor in RNAi. In this study, a plasmid-based system was developed to express dsRNA intracellular from a DNA cassette containing two convergent T7 promoters in Drosophila S2 cells. Efficient knockdown of a transiently expressed reporter gene or an endogenous gene can be achieved by dsRNA expressed from the system. A random cDNA library was constructed in the dsRNA expression cassette and initial screening led to identification of two host factors that are involved in antiviral response in Drosophila S2 cells. The plasmid-based dsRNA expression system provides an alternative tool for functional genomics in Drosophila and other insect cells.
Description: Ph.D. University of Hawaii at Manoa 2014.; Includes bibliographical references.Thu, 01 May 2014 00:00:00 GMThttp://hdl.handle.net/10125/1005512014-05-01T00:00:00ZYang, BaojunIdentification and characterization of f17 : a novel subviral agent that depends on dengue virushttp://hdl.handle.net/10125/100423
Abstract: Dengue is the most important arboviral disease of humans. It is estimated that over 40% of the world's population are at risk of dengue. In recent years, a resurgence of dengue virus has been seen with increased incidences and a wider geographical distribution. Dengue is spread to humans through the bite of an infected female Aedes mosquito. There is currently no vaccine or specific drug treatment for dengue.
In this study, we identified and partially characterized a subviral agent of dengue virus, which we designated as F17. This is the first report of a subviral agent of dengue virus. F17's genome is positive-sense and single-stranded RNA and may encode a capsid protein but the viral particle is unenveloped. F17 was found to replicate efficiently in C6/36 Ae. albopictus and ATC-10 Ae. aegypti cell lines and like many subviral agents, it was found to interfere with its helper virus' replication. In C6/36 cells, the presence of F17 had an enhancing effect and an increase in dengue virus replication was observed. Interestingly, in human U937 DC-SIGN cells, F17 was found to have an inhibitory effect on dengue virus replication. Furthermore, in Vero cells, F17 inhibits dengue virus plaque formation.
This is the first report of a subviral agent of dengue virus and one of a few reported outside of plants. Although arthropod-borne diseases are a major contributor to the burden caused by infectious diseases worldwide, little remains known about the virus-vector-host interactions. The transmission of dengue virus to humans is dependent on a mosquito vector and vector control remains the primary means of preventing vector-borne diseases. F17 was found to increase dengue virus replication in humans and this may have implications in the transmission of dengue virus. The relationship between dengue virus and its subviral agent may also provide insights into the pathogenesis of dengue.
Description: Ph.D. University of Hawaii at Manoa 2014.; Includes bibliographical references.Fri, 01 Aug 2014 00:00:00 GMThttp://hdl.handle.net/10125/1004232014-08-01T00:00:00ZKakinami, Cherie KuuleiFunctional analysis of dengue virus nonstructural protein 1http://hdl.handle.net/10125/100411
Abstract: Dengue virus NS1 is a glycoprotein that plays different roles in different stages of virus life cycle. It exists in multiple forms: intracellular membraneassociated form which is co-localized with dsRNA and essential in viral replication; cell surface-associated form which is associated with lipid raft at the plasma membrane and mediated signaling and complement activation pathways; secreted form which eventually released into the blood stream. In the first part of this study, we investigate the interaction between intracellular form NS1 and dsRNA by EMSA. Our results show that intracellular form NS1 binds dsRNA in vitro and has multiple dsRBDs. Results of the first part implicate a novel role of intracellular form NS1 in viral replication. In the second part of this study, we investigate the binding partner of secreted form NS1. We use affinity pull-down assay to isolate NS1 complex from the supernatant of transfected AD293 cells, and analyze the components by mass spectrometry. We find that ApoA-I, the major structural protein of HDL in serum, is co-immunoprecipitated with NS1. In addition, NS1 is also associated with ApoA-I that is produced and secreted from human HepG2 cells. Delipidation of ApoA-I disrupts its interaction with NS1. Formation of NS1-ApoA-I complex modulates VCAM-1 expression in HMEC-1 cells. Results of the second part indicate a novel role of secreted form NS1 in modulating endothelial cell functions, which may contribute to dengue pathogenesis. In the third part of this study, we investigate the factor that presents in human serum leading to an enhanced DV infectivity in various types of cells. Through co-immunoprecipitation, we reveal that ApoA-I is associated with DV particles and is able to promote DV infection. We further find that siRNA knockdown of SR-BI, the cell receptor of ApoA-I, abolishes the activity of ApoA-I in enhancement of DV infection. FACS analysis of cell surface dengue antigen after virus absorption further confirms that ApoA-I enhanced DV infection via promoting initial attachment of virus to cells. Results of the third part illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.
Description: Ph.D. University of Hawaii at Manoa 2014.; Includes bibliographical references.Fri, 01 Aug 2014 00:00:00 GMThttp://hdl.handle.net/10125/1004112014-08-01T00:00:00ZLi, YujiaRole of hetF and patU3 in the regulation of heterocyst development in Anabaena sp. Strain pcc 7120http://hdl.handle.net/10125/100363
Abstract: A central paradigm in developmental biology concerns the differentiation of cells despite the fundamental sameness of the genetic complement shared by all cells in the organism. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is an ideal model system for the study of development. In response to nitrogen deprivation, Anabaena differentiates nitrogen-fixing heterocyst cells in a periodic pattern. Anabaena research has practical applications in human health, sustainable agriculture, and biofuel production. This study aimed to characterize novel interactions to refine the current understanding of Anabaena development.
Investigations were performed to elucidate the hetF-dependent activation of differentiation. A novel genetic regulatory network involving hetF, a CHF class protease, and the negative regulator patU3 and other developmental genes was identified. A component of the hetZ-patU5-patU3 gene cluster, PatU3 was shown to directly interact with HetZ, another activator of differentiation. Genetic epistasis analysis determined that PatU3 suppressed positive regulation by HetZ and HetR. These negative feedback loops explain the elevated HetR-GFP concentrations in hetF-dependent strains despite the paradoxical absence of heterocysts. The HetF-dependent pathway may act as a control point prior to commitment to the heterocyst cell fate.
Lateral inhibition by PatS and HetN, which both contain the same RGSGR pentapeptide sequence, involves regulation by HetR. The unique domains present in the structure of HetR may relate to its activity as a transcriptional activator. In this study, genetic and cytological approaches were used to identify residues in HetR necessary for interaction with PatS, HetN and RGSGR. A related investigation demonstrated that the RGSGR-pentapeptide derived from HetN directs pattern formation by direct cytoplasmic exchange.
Lastly, protein phosphorylation plays a prominent role in varied biological processes. The PP2C-type protein phosphatase All1758 was characterized in this study. The corresponding all1758 gene is controlled by the developmental genes ntcA and hetR. All1758 affects later stages of differentiation and may represent a critical link between cell growth, cell division and morphogenesis by controlling putative sigma factor regulators (antisigma factors and antisigma factor agonists) and cell division genes (including FtsZ and MinCE). Taken together, these findings support additional regulatory mechanisms necessary for proper Anabaena development.
Description: Ph.D. University of Hawaii at Manoa 2014.; Includes bibliographical references.Fri, 01 Aug 2014 00:00:00 GMThttp://hdl.handle.net/10125/1003632014-08-01T00:00:00ZTom, Sasa K.The HetR regulon of Anabaena sp. strain PCC 7120http://hdl.handle.net/10125/100355
Abstract: The process of cellular differentiation relies on the action of transcriptional regulators to enact the developmental fate of the cell. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a model system for studying pattern formation and cellular differentiation. When combined nitrogen is limiting, Anabaena forms a periodic pattern of nitrogen-fixing heterocyst cells separated by 10-20 photosynthetic vegetative cells. The pattern of cells that can differentiate is defined by the interaction of the master regulator of differentiation, HetR, and the inhibitors of differentiation, PatS and HetN. The crystal structure of HetR bound to DNA determined the amino acids that interact with DNA and mutations in these amino acids yielded non-functional hetR alleles. In this work, a 17 bp inverted repeat in the promoter of the hepA gene was bound by HetR in vitro and found to be necessary for heterocyst-specific transcription in vivo. A search of the genome identified 166 potential hepA-like HetR sites and investigation of a subset of these sites found that HetR could act as either a transcriptional activator or repressor. This study also identified a HetR-binding site in the promoter region of the trpE gene, which encodes an anthranilate synthase involved in tryptophan biosynthesis. Mutation trpE yielded a strain that differentiated heterocysts in the presence of nitrate, a nitrogen source that normally represses differentiation. Analysis of the intracellular concentration of the Kreb's cycle intermediate 2-oxoglutarate (2-OG) showed a roughly 2.5 fold increase within one hour after the transition from growth on ammonia to nitrate. This spike in 2-OG is characteristic of a nitrogen starvation response and suggests a role for amino acid metabolism in the perception of nitrogen starvation. The final study utilized an FMN-dependent fluorphore, EcFbFP, to determine the transcriptional profiles of hetR, patS, and hetN in mature heterocysts. The transcription of hetR and hetN persisted in mature heterocysts while patS expression ceased. This is consistent with roles for PatS in pattern formation, HetN in pattern maintenance, and HetR in all stages of development. This work expands the direct HetR regulon and suggest a role for amino acid metabolism in the induction of differentiation.
Description: Ph.D. University of Hawaii at Manoa 2014.; Includes bibliographical references.Fri, 01 Aug 2014 00:00:00 GMThttp://hdl.handle.net/10125/1003552014-08-01T00:00:00ZVideau, Patrick Jean-AdrienAssessing the Source of Fecal Contamination in Streams on Kaua'i Based on Concentration and Genotypes of FRNA Bacteriophageshttp://hdl.handle.net/10125/22260
Abstract: Extensive data from O'ahu indicate that all streams on this island consistently exceed the USEPA standards (200 fecal coliform/100 ml, 33 enterococci/100 ml) for water quality. Soil was determined to be the source of the elevated counts of these bacteria. In tropical areas, as Hawai'i, these bacteria are able to survive and multiply in the soil. Thus, these bacteria can end up in nearby streams after heavy rains or due to erosion. As a result, the USEPA recommended indicator bacteria (fecal coliform, enterococci) cannot be used to reliably determine when waters in tropical areas are fecally contaminated. Several alternative indicators have been proposed for such areas such as C. perfringens and FRNA coliphages. Extensive monitoring data does not exist for the other islands of Hawai'i. Kaua'i differs from O'ahu in that it is older, wetter and contains an abundance of cesspools. The Nawiliwili Watershed, on the island of Kaua'i, was chosen for this study. Sampling was conducted over a period of one year, and all samples were assayed for the traditional USEPA indicators (fecal, coliform, enterococci) as well as two alternative indicators (C. perfringens, FRNA coliphages). Of the 14 sites sampled, 12 contained levels of fecal coliform and enterococci that exceeded the USEPA standards (200 fecal coliform/100 ml and 33 enterococci/100 ml. This is similar to what has been documented in O'ahu streams. Based on the concentrations of these indicator bacteria, the USEPA would deem these sites as sewage contaminated. However, monitoring of these same sites for C. perfringens indicated that there was no sewage contamination (geometric mean values fell below the proposed standard of 50 CFU/100 ml). FRNA coliphage data indicate that cesspools may be leaching into nearby streams. Two streams (Nawiliwili, Papakōlea) had geometric mean levels greater than the 50 PFU/100 ml (based on O'ahu streams). Other streams in the watershed may be sporadically contaminated by cesspool because elevated FRNA coliphage levels were detected on occasion. Genotyping these FRNA coliphage isolates furthered supported the theory that cesspools were contaminating these sites because 98% of the FRNA isolates were typed as human while only 2% were typed as of animal origin. Current USEPA standards (fecal coliform, enterococci) are not reliable indicators of sewage pollution in tropical areas, thus, alternative indicators such as C. perfringens and FRNA coliphages may prove to be better indicators in these areas.Mon, 01 Aug 2005 00:00:00 GMThttp://hdl.handle.net/10125/222602005-08-01T00:00:00ZVithanage, GayatriAre Fecal Sterols a Possible Alternative Indicator of Human Waste Contamination in Hawaiian Recreational Waters?http://hdl.handle.net/10125/22259
Abstract: Many of Hawaii’s recreational streams and beaches contain high fecal indicator bacteria levels that are not indicative of sewage pollution. Instead, this pollution is due to environmental sources of fecal bacteria which reside and multiply in tropical soils. Current EPA fecal indicator bacteria are no longer representative of human fecal contamination in tropical waters. Fecal sterols have been used as chemical indicators of fecal pollution in many parts of the world. The primary sterol found in human feces is coprostanol. Detection and quantification of coprostanol and related sterols using GCMS analysis provides a fingerprint that can be used to characterize fecal contamination. The objective of this study was to assay for fecal sterols as an independent method to determine whether streams in Hawaii are contaminated with sewage. This method was applied to ambient streams, a stream recently contaminated by a sewage spill, and a stream suspected to be affected by a sewage line leak. The results of this study showed that some ambient streams in Hawaii contain high levels of fecal indicator bacteria, but low concentrations of coprostanol (<10 ng/L). A stream contaminated with sewage during a sewage spill event contained high concentrations of coprostanol (18,000 ng/L) in the first 24 hours after contamination, but this level dropped to ≤ 60 mg/L after 72 hours. A stream suspected to be contaminated with sewage contained significant levels of coprostanol (>1000 ng/L) when fecal indicators were also high, confirming a possible sewage line leak. This study demonstrated that coprostanol is a useful and independent measurement of sewage pollution. It is best used in conjunction with other fecal indicators and human fecal markers if confirmation of human fecal pollution is sought.Mon, 01 Aug 2005 00:00:00 GMThttp://hdl.handle.net/10125/222592005-08-01T00:00:00ZBrostrom, Kathleen A.Assessing the Persistence and Multiplication of Fecal Indicator Bacteria in Hawai'i Soil Environmenthttp://hdl.handle.net/10125/22248
Abstract: Traditional fecal indicator bacteria such as fecal coliform, E.coli and enterococci have been shown to be unreliable indicators of the hygienic quality of recreational waters under tropical conditions. One of the major reasons for considering these bacteria as ineffective indicators of water quality in warm, tropical regions is that they are consistently found in natural environments (plants, soil, water) in the absence of any significant contamination of these environments. Since preliminary studies conducted in Hawaii had indicated soil as the major environmental source of elevated concentrations of these bacteria in environmental waters, the aim of this study was to focus on the soil environment to specifically address two assumptions made by regulatory agencies in using fecal bacteria as indicators of water quality: first, there should not be an environmental source of these indicator bacteria unrelated to sewage or fecal matter contamination, and second, the indicator bacteria do not multiply in the environment. To determine the validity of these two assumptions under tropical conditions in Hawaii and possibly other tropical locations, various experiments were conducted. The major findings are as follows.
1) Analysis of soil samples collected from various locations representing major soil groups on the island of Oahu showed that fecal indicator bacteria are naturally found in most of the soil environments, indicating that the fecal bacteria have adapted to the soil conditions to become part of soil biota. 2) Evidence was obtained to show that the soil contains adequate nutrients to sustain the populations of these bacteria. 3) Growth and multiplication of fecal indicator bacteria in natural soil was dependent on available nutrients (particularly carbon), moisture and competing microorganisms.
In conclusion, tropical soil conditions are suboptimal for the multiplication of fecal indicator bacteria. Consequently, these bacteria in natural soil conditions will probably grow and multiply sporadically when conditions are relatively optimal. Although concentrations of fecal indicator bacteria in soil represent only a small fraction of the microbiota, their counts are significant enough in numbers not only to impact the quality of recreational waters but also to nullify two of the assumptions used in the application of recreational water quality standards. Thus, there is a need for an alternate and more reliable indicator of water quality in Hawaii and other tropical locations.
Description: Includes bibliographical references (leaves 227-244).Fri, 01 Dec 2000 00:00:00 GMThttp://hdl.handle.net/10125/222482000-12-01T00:00:00ZByappananhalli, Muruleedhara N.Effect of bioaugmentation and diesel fuel type on soil bioremediationhttp://hdl.handle.net/10125/21940
Abstract: The enhancement of bioremediation by bioaugmentation in soil contaminated with diesel fuel No. 2 and No. 6 (Bunker C) is uncertain. A clayey soil was contaminated with 6,000 mg of either diesel fuel per kg of soil and seeded (5 x 10-7 cells/g of soil) with a Hawaii soil bacterium (UH138) known to utilize several hydrocarbons. The soil was limed, fertilized, and incubated in jars at 30°C for several months. The concentrations of total petroleum hydrocarbons (TPH) and of polycyclic aromatic hydrocarbons (PAH) in soil were measured by gravimetry and immunoassay, respectively. Poisoned controls (0.6% HgCl2) were used to determine the extent of hydrocarbon degradation due to microbial activity. A rapid first order biodegradation of TPH (84% in 23 days) occurred in soil contaminated with diesel fuel No. 2, regardless of bacterial seeding. Biodegradation of PAH was linear and reached 84% by day 98 in both seeded and unseeded treatments. Bioaugmentation had no effect on bioremediation of diesel fuel No.2. The decrease in TPH and PAH was paralleled by an increase in populations of total bacteria, phenanthrene-degrading bacteria and microorganisms capable of utilizing hexadecane and diesel fuel No. 2 as well as by an enhancement in CO2 evolution by the soil. Indigenous Zygomycetes grew profusely in diesel fuel No. 2 contaminated soil. Cunninghamella echinulata var. echinulata was isolated from the soil and was shown to be able to utilize several hydrocarbons. Thus, Zygomycetes may have contributed to the rapid decrease in contaminant.
In soil contaminated with diesel fuel No. 6, the measurements of TPH and PAH were more variable due to the uneven distribution of the product. No biodegradation of the contaminant occurred over a period of 138 days. The growth of Zygomycetes was scant. The counts of total bacteria remained unchanged after the addition of diesel fuel No. 6. However, counts of the indigenous phenanthrene-degrading bacteria increases dramatically ( 4 log units) during the first 54 days whereas the level of the seeded bacteria remained stable. The counts of mineral oil degraders decrease by 2 log units after day 2. Co2 evolution from the soil confirmed that diesel fuel No. 6 was not degraded by either the indigenous microflora or the seeded bacterium.
Thus, diesel fuel No. 2 was highly degradable by the indigenous population, however, diesel fuel No. 6 was recalcitrant.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 1998.; Includes bibliographical references (leaves 106-117).; Available also on microfiche.Sat, 01 Aug 1998 00:00:00 GMThttp://hdl.handle.net/10125/219401998-08-01T00:00:00ZChua-Chiaco, Barrie WuCharacterization of genes involved in heterocyst differentiation and pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120http://hdl.handle.net/10125/20730
Abstract: The goal of this research was to understand regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120 (hereafter Anabaena PCC 7120) by characterizing regulatory genes for heterocyst formation and their mutants. Anabaena is a filamentous cyanobacterium that forms specialized cells for nitrogen fixation. called heterocysts, which differentiate from vegetative cells at intervals of 10 - 12 cells. Two genes, patS and hetN. are known to suppress the differentiation of vegetative cells into heterocysts for establishing and maintaining a pattern of heterocysts along the filament This study has established that PatS and HetN work independently to suppress differentiation. Using a patS-deletion strain with conditional expression of hetN, it was shown in this study that PatS and HetN are members of separate heterocyst suppression pathways. Inactivation of either of these negative regulators enhances heterocyst frequencies to >20%, compared to 9% in the wild type. However, inactivation of both patS and hetN increases heterocyst differentiation to nearly 100%. A mutant, UHM I 00, was created to study the function of both genes by deleting patS and making expression of hetN conditional. In the absence of nitrogen in liquid medium, almost all vegetative cells of UHM100 differentiated to heterocysts, giving rise to a phenotype called 'multiple contiguous heterocysts' (Mch). Interestingly, UHM100 has an Mch phenotype even in the presence of combined nitrogen, which usually suppresses heterocyst differentiation. UHM100 was used to study the time course of heterocyst differentiation. The percentage of cells that differentiated into heterocysts correlated with the time since induction and was independent of cell density in liquid medium lacking a fixed nitrogen source. It was expected that in the patS and hetN double mutant UHM100, hetR will be overexpressed in all cells. However, when UHM100 containing hetR-gfp was grown in nitrogen-free medium, the pattern of fluorescence observed at 48 h after induction showed that hetR was expressed in ~55% cells, suggesting that nitrogen-deprivation did not immediately induce hetR in all cells. Thus, hetR expression in the absence of patS and hetN was asynchronous. The patS and hetN double mutant was also used to determine if the position of heterocysts along the filament was random or not When the positions of heterocysts relative to other vegetative cells was examined using statistical analyses for randomness, the distribution of heterocysts was found to be nonrandom. Time course studies using UHM100 further showed that heterocyst differentiation occurred in clusters of 2-5 cells at 48 h and the size of the clusters increased with time. Heterocyst frequency reached -98% after 144 h in the absence of fixed nitrogen. Clustering of heterocysts in the filaments of UHM100 suggests that besides PatS and HetN, there are other factors that influence pattern formation in Anabaena PCC 7120. A heterocyst-deficient (Her) spontaneous mutant, NSM6, was isolated from UHM100. Complementation of NSM6 by a cosmid clone, pPB6-1, from an Anabaena PCC 7120 genomic library restored the Mch phenotype of this mutant. By sequencing, sub-cloning, and further complementation analyses, a novel gene, alr9018, was identified in a 4.l-kb fragment of pPB6-1. The alr9018 gene is located in the Epsilon plasmid of Anabaena, and it encodes a 148.7-kDa protein. The Alr9018 protein contains an NTPase domain, which is a characteristic of proteins involved in signal transduction. alr9018 is expressed in both vegetative cells and heterocysts. Similar to alr9018, hetR can also restore the Mch phenotype in NSM6, suggesting that the NSM6 mutant can be functionally complemented by multiple copies of either a/r9018 or hetR. When palr9018 was transferred to Anabaena PCC 7120, the transconjugants formed ~15% heterocysts compared to ~10% heterocysts formed by Anabaena PCC 7120. The transconjugants also reduced at least 50% more acetylene than PCC 7120, suggesting that multiple copies of alr9018 enhance heterocyst development. This is the first report showing that the Epsilon plasmid of Anabaena PCC 7120 contains genes involved in heterocyst differentiation. The identification and characterization of alr9018 in the present study further show that the regulation of heterocyst differentiation in Anabaena is complex. Further studies will be required to fully understand the complex interactions between alr9018 and hetR and the role of alr9018 in cell differentiation and pattern formation in Anabaena PCC 7120.
Description: Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; The goal of this research was to understand regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120 by characterizing regulatory genes for heterocyst formation and their mutants. Anabaena is a filamentous cyanobacterium that forms specialized cells for nitrogen fixation, called heterocysts, which differentiate from vegetative cells at intervals of 10--12 cells. Two genes, patS and hetN, are known to suppress the differentiation of vegetative cells into heterocysts for establishing a de novo pattern and maintaining a pattern of heterocysts along the filament. A mutant, UHM100, was created to study the function of both genes by deleting patS and making expression of hetN conditional. This study has established that PatS and HetN are members of two separate heterocyst suppression pathways. In absence of nitrogen, inactivation of both patS and hetN increases heterocyst differentiation to nearly 100%, giving rise to a phenotype called 'multiple contiguous heterocysts' (Mch). UHM100 has an Mch phenotype even in the presence of combined nitrogen, which usually suppresses heterocyst differentiation. In absence of both patS and hetN, the expression of hetR, a master regulator of heterocyst differentiation, was observed in &sim;55% cells and was asynchronous. The distribution of heterocysts next to a vegetative cell in UHM 100 was found to be nonrandom. These results suggest that besides PatS and HetN, there are other factors that influence pattern formation in Anabaena PCC 7120. A heterocyst-deficient (Hef) spontaneous mutant, NSM6, was isolated from UHM 100. A novel gene, alr9018, from the Anabaena Epsilon plasmid complemented NSM6 and restored the Mch phenotype of this mutant. Transconjugants of Anabaena PCC 7120 containing the cloned alr9018 gene fixed 50% more N2 than PCC 7120, suggesting that multiple copies of alr9018 enhance heterocyst development. This is the first report showing that the Epsilon plasmid of Anabaena PCC 7120 contains genes involved in heterocyst differentiation. Expression of alr9018 was observed in both vegetative cells and heterocysts. Similar to alr9018, hetR could also restore the Mch phenotype in NSM6, suggesting functional similarity between a1r9018 and hetR. The Alr9018 protein contains an NTPase domain, which is a characteristic of proteins involved in signal transduction.; Includes bibliographical references (leaves 89-97).; Also available by subscription via World Wide Web; 97 leaves, bound 29 cmTue, 01 Jan 2008 00:00:00 GMThttp://hdl.handle.net/10125/207302008-01-01T00:00:00ZBorthakur, Pritty B.Bacterial profiles in healthy and Montipora white syndrome affected Montipora capitata mucus and the identification of potential etiologic agentshttp://hdl.handle.net/10125/20729
Abstract: Montipora white syndrome (MWS) is a progressive tissue loss disease that affects Montipora capitata, a major reef building coral in Kaneohe Bay, Oahu. Chronic MWS manifests as focal to multifocal variably-sized areas of tissue loss, revealing an intact white skeleton bordered by normal appearing tissue. Acute MWS progresses faster than chronic and manifests as a large solitary area of tissue loss revealing intact white skeleton bordered by pale tissue that transitions into normally colored tissue. Culture-dependant methods were used to determine the bacterial community structure associated with healthy and MWS affected M capitata mucus. Healthy mucus contained 5.59 x 102 CFU/ml of culturable bacteria that was predominantly Alteromonas and Streptomyces. MWS affected mucus samples had an average of 25.6 times higher bacterial load at 1.42 x 104 CFU/ml of mucus. The culturable bacterial community structure of mucus from diseased coral was primarily composed of Vibrio spp., in particular, V. harveyi. The second most abundant bacteria isolated from mucus was Pseudoalteromonas and Ruegeria in acute and chronic MWS, respectively. Based on these bacterial profiles, Alteromonas, V. harveyi, Pseudoalteromonas and Ruegeria isolates were selected for in vitro challenge experiments. Alteromonas was used as a negative bacterial control because it is commonly found in abundance in healthy M capitata mucus. Preliminary challenge experiments eliminated V. harveyi and Ruegeria as etiologic agents. Though no tissue loss was observed, mucus after inoculation with Ruegeria had a slightly elevated CFU/ml. Fragments exposed to Pseudoalteromonas had varying responses to the challenge. Tissue loss, thinning and, alternatively, no apparent signs of deteriorating health were all observed after the challenge with Pseudoalteromonas. Overall. CFU/ml of mucus was increased post challenge with Pseudoalteromonas for all three observations. In future studies, additional challenge replicates should be conducted to further investigate the role of Pseudoalteromonas in coral pathogenesis.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2008.; Includes bibliographical references (leaves 59-69).; ix, 87 leaves, bound ill. 29 cmTue, 01 Jan 2008 00:00:00 GMThttp://hdl.handle.net/10125/207292008-01-01T00:00:00ZSmith, AshleyResearch on the diversity and the biomedical potential of marine fungi in Hawaiihttp://hdl.handle.net/10125/20728
Abstract: This study was designed to survey the diversity of marine fungi in Hawaiian waters using molecular biological methods and to do preliminary screening for biological isolates with pharmaceutical potential based on biological activities. 106 marine fungal isolates were isolated from nine algae species from eight different locations around the coast of the Hawaii Island in Hawaii. These fungal isolates were classified based on the ribosomal internal transcribed spacer regions, and were found to represent 10 orders including 57 species of Ascomycota, and 2 orders including 3 species of Basidiomycota. Two isolates, Haw 3BII and Haw 7A3, representing long phylogenetic distances from known sequences in Genbank, are potential new species. Anti-bacterial assays were performed with the crude extracts obtained from the cultures of 70 algae associate fungi in Hawaii. In total, eight extracts shown inhibitory activity to Bacillus subtilis and four to Staphylococcus aureus, respectively. None inhibited activity to the gram negative bacteria Pseudomonas aeruginosa and E.coli K12. Anti-tumoral assays using murine cell lines BCN (non-tumorgenic) and L88 (tumorgenic) were performed with the crude extracts of the cultures of 70 algae associated fungi and 58 sponge-associated fungi. In total, four extracts, one from algae- associated fungi and three from sponge-associated fungi, showed strong inhibition of L88 and slight or no inhibition of BCN. Those isolates will be further studied for their potential in drug discovery.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2007.; Includes bibliographical references (leaves 50-54).; x, 54 leaves, bound ill. 29 cmMon, 01 Jan 2007 00:00:00 GMThttp://hdl.handle.net/10125/207282007-01-01T00:00:00ZHe, MingxiaoGenes involved in nitrogen fixation and heterocyst differentiation in Anabaena sp. PCC 7120http://hdl.handle.net/10125/20727
Abstract: Cyanobacteria, also commonly known as blue green algae, emerged approximately 3.5 billion years ago as the first photosynthetic prokaryotes on Earth. Accumulation of oxygen generated as a byproduct of their photosynthetic activity changed the nature of the primitive atmosphere from anoxic to oxic, leading to the evolutionary transition that resulted in the emergence of aerobic organisms and consequently in the rise of higher plants and animals that predominate the ecosystems in today's biosphere. In addition to generation of oxygen, cyanobacteria have also been of considerable importance for their role in fixing atmospheric dinitrogen to produce other forms of nitrogen, such as ammonium, that is utilizable to other organisms. Many of the free-living and symbiotic cyanobacteria contribute significantly in fertilizing aquatic and terrestrial environments with their ability of nitrogen fixation, sustaining growth and prosperity of a wide range of populations in those habitats. Study of cyanobacteria, therefore, has been an intensive area for their significant ecological roles in various ecosystems. Perhaps of equally important reason for the growing popularity of studying cyanobacteria comes from the fact that they are the first filamentous multicellular organisms that appeared on Earth. In addition, they also exhibited cellular differentiation to form heterocysts, cells specialized for nitrogen fixation. Production of oxygen caused by photosynthetic activity of cyanobacteria imposed a problem on nitrogen fixation, which is mediated by nitrogenase, an enzyme extremely sensitive to oxygen. Evolution of oxygen would inevitably inactivate nitrogenase, with both oxygen and nitrogenase being present intracellularly. Some species of cyanobacteria evolved a temporal separation of photosynthesis and nitrogen fixation, carrying out the former during the day and the latter at night Some other species, such as Anabaena, produced differentiated cells called heterocysts, which do not perform the oxygen-evolving part of photosynthesis and form extra layers of an envelope to prevent oxygen diffusing into the cell, creating an ideal microaerophillic environment for nitrogenase activity. A fixed form of nitrogen produced in heterocysts in a filament then is distributed to the photosynthesizing cells so the filament as a whole does not starve for nitrogen while still capable of photosynthesis. For efficient distribution of a fixed source of nitrogen from heterocysts to photosynthesizing cells, or more commonly called vegetative cells, heterocysts are evenly spaced in filaments. The genera of cyanobacteria forming the two distinct types of cells demonstrate a one-dimensional pattern of multicellularity between the two different cell types, providing the simplest example of pattern formation and developmental regulation. A series of technological breakthroughs in molecular biology in the past decades have been enabling approaches on molecular and genetic levels to elucidate complex mechanisms governing cellular differentiation and pattern formation in some species of cyanobacteria. In this study, mechanisms governing nitrogen fixation, cellular differentiation, and pattern regulation in Anabaena sp. PCC 7120, a filamentous cyanobacterium capable of forming heterocysts in a regulated pattern, were examined and reported.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 45-57).; vi, 57 leaves, bound ill. 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207272006-01-01T00:00:00ZYamaura, HiroshiGenes involved in diazotrophic growth of Anabaena sp. PCC 7120http://hdl.handle.net/10125/20726
Abstract: In Anabaena, in the absence of fixed form of nitrogen, vegetative cells differentiate into nitrogen-fixing heterocysts at semiregular intervals along the filament. Heterocysts can be distinguished from vegetative cells microscopically by their larger size and thicker cell envelope. In order to create microaerophilic conditions for the activity of nitrogenase, they have two additional layers of envelope made of polysaccharides and glycolipids (34). The glycolipid layer, which is the innermost of the two, provides a hydrophobic barrier against the entry of oxygen (46, 48). The exterior polysaccharide layer is thought to preserve the integrity of the glycolipid layer. Genes necessary for the production and loca1ization of both layers have been found and pathways for their synthesis have been proposed (13, 20). Once a microaerophilic environment has been created inside the heterocysts, they fix atmospheric nitrogen and transport it to vegetative cells and in return receive a source of reductant required for fixation from vegetative cells (42). The pattern of heterocysts along a filament is determined by the interplay of positive and negative acting regulatory factors, and approximately 12 hours after the removal of fixed nitrogen, select cells have committed to termina1 differentiation into heterocysts (31, 50, 56). PatS and HetR appear to be the two central factors that control differentiation and pattern formation. HetR is the master regulator and has both DNA-binding and protease activity (7, 21, 57). It displays positive autoreglation and expression of hetR is induced in proheterocysts prior to commitment to differentiation (5). In order to identify the genes involved in diazotrophic growth and differentiation of heterocysts by Anabaena, a genetic screen was conducted to isolate mutants incapable of growth in the absence of fixed nitrogen. Interruption of the coding region of fraG, the gene upstream of hetR, by a transposon resulted in a fragmentation mutant that was unable to grow in the absence of a fixed source of nitrogen. The predicted protein is similar to permeases and is necessary for filament integrity and maturation of heterocysts to the point of glycolipid layer formation. The enzymes that synthesize and remodel the peptidoglycan are generally known as penicillin binding proteins (PBPs). In E. coli, 12 PBPs have so far been identified, of which only PBPs la and 1b are essential for cell viability (54). In Anabaena, peptidoglycan is present inner to the glycolipid layer. A pbpB mutant of Anabaena was incapable of fixing atmospheric nitrogen under aerobic conditions (28). This mutant in the presence of fixed nitrogen did not show any significant difference in the phenotype with respect to that of the wild type. But, in the absence of a fixed form of nitrogen, filaments were yellow, short and twisted. Vegetative cells were unequal in size and shape. Heterocysts were distorted with thin envelopes and with no cyanophycin granules at the poles. In the genetic screen described, a pbp6 mutant that was unable to fix atmospheric nitrogen under aerobic conditions was obtained. The predicted protein consists of both the transglycosidase as well as 1ranspeptidase domains that might be involved in the formation of peptidoglycan in Anabaena.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 47-55).; vi, 55 leaves, bound ill. 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207262006-01-01T00:00:00ZNayar, Asha SivasankaranKauai's potable groundwater sources : assessing its vulnerability to fecal contamination for the pending groundwater rulehttp://hdl.handle.net/10125/20725
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 105-109).; ix, 109 leaves, bound col. ill., col. maps 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207252006-01-01T00:00:00ZSato, DaynaImpact of tropical plants on microbial activity and diversity in soil contaminated with petroleum hydrocarbonshttp://hdl.handle.net/10125/20724
Abstract: The effect of plants (milo, oleander and buffelgrass) and a hexadecane and phenanthrene mixture (1 g and 200 mg/kg soil, respectively) on the diversity and activity of hydrocarbon-degrading bacteria in a sandy coastal soil was investigated. Two/third of hexadecane was degraded after 56 days. Hydrocarbon depletion was not plant-enhanced but was retarded slightly by milo and buffelgrass. Lipase activity, an a1kane-metabolism indicator, increased during rapid hexadecane depletion (days 0-56). The diversity of the dominant hexadecane-degrading bacteria was based on partial sequencing of 168 rDNA. On day 0, mainly Alphaproteobacteria were found By day 56, Gammaproteobacteria dominated the contaminated samples whereas similar numbers of Alphaproteobacteria and Gammaproteobacteria genotypes dominated the uncontaminated samples. Alcanivorax was found in all contaminated samples except for buffelgrass rhizospheres, which harbored only Pseudomonas sp. IMT40. With little hexadecane left by day 114, similar abundances of Alphaproteobacteria and Gammaproteobacteria genotypes occurred in all samples. Alcanivorax had virtually disappeared.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 138-163).; xii, 163 leaves, bound ill. 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207242006-01-01T00:00:00ZShibata, Alexandra KuCharacterization of secretogranin III in mast cellshttp://hdl.handle.net/10125/20723
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 84-91).; ix, 91 leaves, bound ill. 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207232006-01-01T00:00:00ZPrasad, PrernaInvestigating the regulation of fatty acid degradation in Pseudomonas aeruginosahttp://hdl.handle.net/10125/20722
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 143-160).; xx, 160 leaves, bound ill. 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207222006-01-01T00:00:00ZNguyen, David TranCharacterization of the Acyl-CoA binding domain containing 3 proteinhttp://hdl.handle.net/10125/20721
Abstract: The cAMP dependent protein kinase, PKA, is of great importance for cells to transduce extra- and intra-cellular signals. Recent studies have found that signal transduction through PKA is coordinated by a group of scaffold proteins caned A-Kinase Anchoring Proteins. These AKAPs, together with PKA and its substrates, form a dynamic assembly that tightly controls the location and timing of signal transduction events, which in turn regulates various cellular activities including ion channel modulation, cell growth, cell differentiation and cytokinesis. Previous research in Dr. Turner's lab has found that TRPY ion channels recruit PKA through Acyl-CoA Binding Domain containing 3 protein (ACBD3) to transduce physiological stimuli in mast cells and sensory neurons. Other studies have indicated a similar role for ACBD3 during PKA-mediated steroid formation. Hence, we hypothesize that ACBD3 may function as an AKAP recruiting PKA to multiple novel targets, in addition to ion channels such as the TRPVs. The project presented in this paper aimed at production of purified ACBD3 protein, for use as an affinity matrix to purify novel targets of ACBD3. The human ACBD3 cDNA was subcloned into the pTrcHisB vector with a conferred epitope tag comprising six sequential Histidine residues. Then the construct was transformed into the BL21 E.coli strain to express the protein in an inducible manner. Finally, we used Immunobilized Metal Affinity Chromatography (IMAC) technique to purify the fusion protein. With the purified protein, we confirmed the strong PKA binding ability of ACBD3, which is consistent with our hypothesis. We also demonstrated a wide expression profile of ACBD3 in different tissues, suggesting that ACBD3 is likely to function outside the CNS and hence is likely to interact with proteins other than TRP The cAMP dependent protein kinase, PKA, is of great importance for cells to transduce extra- and intra-cellular signals. Recent studies have found that signal transduction through PKA is coordinated by a group of scaffold proteins caned A-Kinase Anchoring Proteins. These AKAPs, together with PKA and its substrates, form a dynamic assembly that tightly controls the location and timing of signal transduction events, which in turn regulates various cellular activities including ion channel modulation, cell growth, cell differentiation and cytokinesis. Previous research in Dr. Turner's lab has found that TRPY ion channels recruit PKA through Acyl-CoA Binding Domain containing 3 protein (ACBD3) to transduce physiological stimuli in mast cells and sensory neurons. Other studies have indicated a similar role for ACBD3 during PKA-mediated steroid formation. Hence, we hypothesize that ACBD3 may function as an AKAP recruiting PKA to multiple novel targets, in addition to ion channels such as the TRPVs. The project presented in this paper aimed at production of purified ACBD3 protein, for use as an affinity matrix to purify novel targets of ACBD3. The human ACBD3 cDNA was subcloned into the pTrcHisB vector with a conferred epitope tag comprising six sequential Histidine residues. Then the construct was transformed into the BL21 E.coli strain to express the protein in an inducible manner. Finally, we used Immunobilized Metal Affinity Chromatography (IMAC) technique to purify the fusion protein. With the purified protein, we confirmed the strong PKA binding ability of ACBD3, which is consistent with our hypothesis. We also demonstrated a wide expression profile of ACBD3 in different tissues, suggesting that ACBD3 is likely to function outside the CNS and hence is likely to interact with proteins other than TRPVs.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2006.; Includes bibliographical references (leaves 53-55).; ii, 55 leaves, bound ill. (some col.) 29 cmSun, 01 Jan 2006 00:00:00 GMThttp://hdl.handle.net/10125/207212006-01-01T00:00:00ZLi, ZengqiuCytotoxicity and antiproliferative effects of extracts from coffee cherry fruit on cell lines from normal breast tissue and from non-invasive and invasive breast cancershttp://hdl.handle.net/10125/20720
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2005.; Includes bibliographical references (leaves 106-109).; xxi, 109 leaves, bound ill. (some col.) 29 cmSat, 01 Jan 2005 00:00:00 GMThttp://hdl.handle.net/10125/207202005-01-01T00:00:00ZMeujo, Damaris AgatheThe effects of acute infection and inflammation on phase II detoxifying enzymeshttp://hdl.handle.net/10125/20719
Abstract: Infection and inflammation may alter liver metabolism causing significant changes in drug efficacy and toxicity. The expression and activity of the Cytochromes P450 in human liver are significantly down-regulated during acute infection and inflammation but little is known about Phase II metabolizing enzymes. We treated the human liver cell line HepG2 with TNF-α and IL-1β to at 0.1-1000 U/mL for 0-48 hours as a model of acute infection and inflammation. Cells were harvested at each time point and assessed for cell death (MIT assay) and levels of reactive oxygen species (ROS) and for the expression and activity of the major Phase II enzymes UDP-glucuronosyl transferase (UGT), Glutathione-S-transferase (GST) and Sulfotransferase (SULT). Since all of these enzymes have hepatic nuclear factor (HNF) recognition sequences in their genes, we assessed the effects of the cyrokines on HNF1 and HNF4 expression and nuclear translocation with immunofluorescence. UGT enzymes are not significantly affected by pro-inflammatory cytokines and SULT enzymes showed no change in activity while GST enzymes showed a significant increase in activity but returned to norma1levels by 48 hours. Higher GST activity may confer lower efficacy of drugs during acute infection and inflammation.
Description: Thesis (M.S.)--University of Hawaii at Manoa, 2007.; Includes bibliographical references (leaves 71-82).; xiii, 82 leaves, bound 29 cmMon, 01 Jan 2007 00:00:00 GMThttp://hdl.handle.net/10125/207192007-01-01T00:00:00ZWelch, Darcy L.Pseudomonas aeruginosa pathogenesis during cystic fibrosis lung infection and metabolism of lung surfactant component phosphatidylcholinehttp://hdl.handle.net/10125/20718
Description: Includes supplementary digital materials.; Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is the principal cause of hospital-acquired pneumonia, and is responsible for the high morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa also causes a wide range of human diseases including otitis media, keratitis, endocarditis, osteochondritis, pyelonephritis, cellulites and septicemia. Despite the considerable effort expended toward studying P. aeruginosa infections and virulence expression, the pathogenesis of P. aeruginosa-associated diseases and the ability of P. aeruginosa to reach high cell density (HCD) during chronic lung infections in CF patients remains enigmatic. We hypothesized that in the lung environment, P. aeruginosa degrades the naturally occurring lung surfactant component phosphatidylcholine (PC), as a nutrient source to afford HCD replication and maintenance.; To test this central hypothesis, I have conducted microarray and real-time RT-PCR experiments directly on sputum samples from two chronically infected CF patients to obtain a snap shot of the metabolic profile of P. aeruginosa populations. The microarray and real-time RT-PCR data provide strong evidence substantiating our initial hypothesis that PC may serve as a nutrient source to afford HCD replication and maintenance.; Using bioinformatics and the microarray data, I was able to identify the PC degradative genes expressed during P. aeruginosa lung infection, including the fatty acid degradative (fad), choline metabolism (bet), and glycerol metabolism (glp) genes. The bet and glp pathways have been previously characterized, however, the fad pathway remains a mystery and can only be predicted based on the established E. coli fad pathway. Using this pathway as a model, I initiated investigations into deciphering this pathway in P. aeruginosa. I present here the characterization of the fadD genes (acyl-CoA synthetases), preliminary data on the characterization of the fadBA operons (acyl-CoA thiolases and acyl-CoA dehydrogenases), and data that link the association of the fad, bet, and glp pathways in regard to PC and PC component degradation.; Includes bibliographical references (leaves xxx-xxx).; Also available by subscription via World Wide Web; 293 leaves, bound 29 cmTue, 01 Jan 2008 00:00:00 GMThttp://hdl.handle.net/10125/207182008-01-01T00:00:00ZSon, Mike SeongBase composition of Mycoplasma DNA and the formation of specific DNA-DNA hybridshttp://hdl.handle.net/10125/11753
Abstract: Native deoxyribonucleic acid (DNA) was extracted and purified from eight parasitic Mycoplasma species and two saprophytic strains of Mycoplasma laidlawii. Triplicate samples of each of the DNA types were subjected to thermal denaturation (melting) to estimate the mean mole percent guanine plus cytosine (G+C%) composition. The DNA melting transitions were followed spectrophotometrically, where the relative absorbance was plotted as a function of temperature. From the midpoints of each transition the Tm (midpoint of the hyperchromic shift) values were estimated and the G+C% calculated from the following relationship: Tm = 69.3 + 0.41 (G+C). These experiments showed that the base composition of both the saprophytic and the parasitic Mycoplasma DNAs were characterized by a narrow G+C range of 32% to 35%. with the exception, of two species: Mycoplasma gallinarum, an avian parasite, 28% G+C; and the murine parasite Mycoplasma arthritidis, 29% G+C. Despite the narrow compositional range, the regions of greatest inflection of the melting transitions, from 17% to 85% of the hyperchromic shifts, were variable and indicated a compositional heterogeneity. A mathematical analysis of the transitions estimated the extent of the compositional heterogeneity in the different DNA types. Experiments were designed to evaluate the kinetics of immobilization of DNA to nitrocellulose membrane filters. Filtration of high molecular weight, denatured DNA, onto membranes resulted in the irreversible immobilization of the DNA to the membranes. DNA which was shear degraded by sonication for varying periods of time, then denatured, was bound irreversibly to the membranes. Incubation of blank membranes with an albumin solution and labeled sheared-denatured DNA, showed that the DNA was nonspecifically adsorbed to the membranes at about 10% of the input DNA concentration. The adsorption decreased slightly as the duration of exposure to shearing increased. Washing the membranes after incubation reduced this adsorption to about 2% of the input DNA. When sheared and denatured DNA was incubated with membranes in the absence of the albumin solution, the nonspecific adsorption was about twice that detected than in its presence. After washing, a similar 2% of the input DNA remained permanently adsorbed to the membranes. E. coli DNA immobilized on membranes was incubated with homologous labeled DNA to determine saturation of hybridizing sites as a function of the ratio of labeled to total DNA (symbolized as T.). When the value of T was about 0.5, nearly all of the labeled DNA was bound to the immobilized DNA. When the value of T was greater than 0.5, retention of DNA to the membranes decreased. This implied that all the hybridization sites of the immobilized DNA were saturated according to the expectation of the binomial distribution. Hybridization of homologous and heterologous Mycoplasma DNA by a modification of the nitrocellulose membrane filter technique was employed to study genetic relatedness among the Mycoplasma. Radiophosphorus labeled DNA from saprophytic Mycoplasma was bound to immobilized parasitic Mycoplasma DNA in all cases except for M. arthritidis. Labeled DNAs from E. coli K-12 and s. marcescens did not hybridize with Mycoplasma DNA, but did cross react with each other. Relative DNA-DNA homologies indicated that about 20 to 130 common cistrons were shared by the saprophytic and the parasitic Mycoplasma DNAs. Labeled DNA from B. circulans in similar hybridizations with Mycoplasma DNA indicated about 170 to 250 mutual cistrons. Hybridizations between B. circulans and Mycoplasma DNAs occurred only when the DNA from the Mycoplasma was immobilized to membranes. In reciprocal hybridizations no detectable homologies were observed. The possibility that the DNA extracted from B. circulans was contaminated by and complexed with teichoic acid was discussed as an explanation for the absence of reciprocity. This argument proposed that the retention of B. circulans DNA to DNA-membranes was not genuine, but rather a reflection of spurious adsorption of the teichoic acid. Based on the agreement between the relative amount of binding in reciprocal combinations of the saprophytic strain A and B DNA, it was concluded that these Mycoplasma strains are closely related. Genetic homology of some common cistrons also exist between the saprophytic and most of the parasitic Mycoplasma DNAs examined. Genetic relatedness between the DNAs from B. circulans and from Mycoplasma was concluded not to be definitive.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1967.; Bibliography: leaves 77-86.; ix, 86 l graphs, tablesSun, 01 Jan 1967 00:00:00 GMThttp://hdl.handle.net/10125/117531967-01-01T00:00:00ZFox, Richard HowardStructural and genetic studies of chicken 7S immunoglobulin allotypeshttp://hdl.handle.net/10125/11752
Abstract: Six chicken 7S immunoglobulin (Ig) allotypic specificities were demonstrated with antisera produced in highly inbred chickens. A radioimmunoassay which utilized intact, radiolabeled 7S Ig alloantigens was developed, and was used for the structural and genetic analysis of these specificities. The binding of dinitrophenylated alloantiserum (DAA) with radiolabeled alloantigen was demonstrated by the addition of rabbit anti-dinitrophenyl antiserum followed by precipitation of complexes with sheep anti-rabbit γ-globulin antiserum. Binding inhibition assays with the DAA method detected allotypes on 2 to 5 ng of 7S Ig alloantigen. Structural analyses of the six allotypic specificities were performed by direct binding and binding inhibition analysis with the DAA assay. Specificities CS-1.1 and CS-1.2 were present on papain-produced Fab fragments and isolated 7S Ig heavy (H) chains; but not on light (L) chains, Fc fragments, or 17S Ig. For both specificities H chains reassociated with L chains were three- to five-fold better inhibitors on a weight basis than isolated H chains. Regression coefficients calculated from binding inhibition curves indicated that the avidity of H chains for specific alloantisera was significantly increased following reassociation with L chains, suggesting that the conformation of CS-1.1 and CS-1.2 determinants may depend in part upon Hand L chain interactions. Specificities CS-1.3 and CS-1.4 were detected on isolated 7S Ig H chains; but not on L chains, Fab, Fc, or l7S Ig. In contrast to the results with CS-1.1 and CS-1.2, reassociation of Hand L chains did not significantly increase the inhibitory activity or binding avidity of CS-1.3 and CS-1.4 for specific alloantisera. Specificity CS-1.5 was also present on intact 7S Ig, but absent from Fab, Fc, or 178 Ig. A nonhomologous cross reaction was observed in binding inhibition assays with UCD 2 anti-CS-1.2 alloantiserum. The cross-reacting specificity, designated CS-1.6, was found on 7S Ig and was absent from l7S Ig enriched preparations. These data indicate that at least two regions of the chicken 7S Ig H chain contain genetic variations: (1) specificities CS-1.1 and CS-1.2 are located in the Fd portion of the H chain, perhaps in a region analogous to the CH1 domain of mammalian IgG; and (2) specificities CS-1.3, CS-1.4, and CS-1.5 are located in a region of the H chain which is sensitive to papain digestion. Among F2 progeny, these specificities segregated in a manner statistically indistinguishable from that expected of codominant alleles of a "Mendelian" gene at an autosomal locus. This gene, designated CS-1, occupied a locus which was not closely linked to any of five chicken blood group loci. Combinations of 7S Ig allotypic specificities were inherited as stable phenogroups among the F2 progeny of inbred chicken lines, and direct binding analysis with the DAA assay indicated that the specificities forming phenogroups were present on the same 7S Ig molecules. These results also demonstrated that more than 94% of the serum 7S Ig contained the products of the CS-1 gene. In FI hybrids with the genotype CS-1.1,1.3/1.2, two populations of serum 7S Ig molecules were detected by direct and sequential binding analysis with alloantisera. One population of 7S Ig contained specificities CS-1.1 and CS-1.3, but not CS-1.2; while the second population carried only specificity CS-1.2. Therefore, each population was exclusively the product of one parental allele. Consistent with a genetic regulatory mechanism involving allelic exclusion, no 7S Ig containing allotypes produced by both alleles was detected. A survey of 43 inbred lines derived from four sources in the United States for 7S Ig allotypes revealed considerable genetic polymorphism. A minimum of ten alleles of the CS-1 gene were found as unique combinations of CS-1 specificities. A system of nomenclature for CS-1 alleles was developed and six inbred lines were designated as prototype lines. The remaining four CS-1 alleles occurred only in inbred lines which were polymorphic for 7S Ig allotypic specificities.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii at Manoa, 1976.; Bibliography: leaves 141-150.; Microfiche.; xv, 150 leaves illThu, 01 Jan 1976 00:00:00 GMThttp://hdl.handle.net/10125/117521976-01-01T00:00:00ZWakeland, Edward K.Factors affecting 7S and 17S antibody concentrations and affinities in chickenshttp://hdl.handle.net/10125/11751
Abstract: The formation of different immunoglobin classes and binding constants were determined during the immune response in chickens injected with highly substituted dinitrophenyl-bovine gamma globulin (DNP-BGG). A comparison between intravenous immunization and intramuscular injections with adjuvants was made. Antisera were assessed for hemagglutination and precipitating anti-DNP antibody, and the presence of 78 and 178 antibodies was detected by radioinmunoe1ectrophoresis (RIE). The binding constants were measured from 78 and 17S globulin preparations using extensive equilibrium dialysis determinations. Following an intravenous injection of DNP-BGG, the primary response was characterized by a transient synthesis of low amounts of 7S and 178 anti-INP antibodies. A single intravenous injection did not induce sufficient amounts of antibody to perform equilibrium dialysis studies. However, a second intravenous injection resulted in a more vigorous production of both classes of antibodies. Hem agglutinating titers were high and precipitins were present within 4 days after the second injection. Furthermore, the 178 antibody was detected for at least 4 months by RIE. In contrast to the response to intravenous injections, the response of chickens given a single intranus0l1ar injection of antigen in either Freund's complete (FCA) or incomplete (FIA) adjuvants was characterized by an initial synthesis of 78 and 178 antibodies followed by the exc1usive and persistent production of 7S antibodies. Hemagglutinating titers were low even after 2 intramuscular injections of antigen in adjuvant. After a single injection of antigen in FCA with doses ranging from 0.02 to 20 mg, binding of the 7S anti-DNP antibody was detected by RIE for at least 490 days. As a result of either intravenous or intramuscular injections of INP-BGG, the anti-BGG responses were more transient in nature than the anti-hapten responses. After two intravenous injections little or no anti -BGG antibodies were detected by RIE one month later. Persistent synthesis of 7S anti-BGG antibodies generally required 2 intramuscular injections of DNP-BGG in FCA. Injections of low doses of antigen in either FCA or FIA in chickens induced 7S antibodies of high affinity (-ΔF° > 10 kcal/mole) by 3 months. The non-linearity of the Sips plots generated from the binding data indicated that a shift in the distribution of antibody affinities occurred due to the production of high affinity antibodies. In birds immunized with FCA the high affinity antibodies had restricted heterogeneity as indicated by heterogeneity indices of 1. Chickens, given multiple intravenous injections, however, failed to produce high affinity antibodies. Despite altering the priming dose, the interval between injections and the number of injections, chickens injected intravenously produced 7S antibodies with -ΔF° values that were consistently less than 10 kcal/mole. Sips plots of the binding data indicated that the antibody affinities were distributed in a normal array. It appears that at least two important conditions are required for eliciting high affinity antibody, namely, limited concentrations of antigen and the adjuvant effects. Nearly identical affinities and heterogeneity indices were obtained for both the 7S and 17S antibodies isolated from 12 individual chickens given 2 intravenous injections of antigen. Thus, intravenous injections of antigen failed to elicit high affinity 7S and 17S antibodies. The conditions required to generate circulating 17S antibody of high affinity are not known. In chickens given 2 intravenous injections of DNP-BGG, more 7S antibody was produced when the interval between intravenous injections was short than when the interval was longer. Intravenous injections apparently failed to induce large numbers of long-lived memory cells. Less antibody was synthesized in birds given either low (0.02 mg) or high (20 mg) doses of priming antigen than in birds injected with intermediate doses. Specifically purified low affinity 7S antibodies had valences of less than 2, whereas a valence close to 2 was found for antibodies of higher affinities. Similarly l7S antibody of low affinity gave valences less than 10. Obtaining valences less than the expected value is best explained on the basis of contamination of the preparation with nonbinding proteins or the presence of antibody with very low affinity.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii at Manoa, 1974.; Bibliography: leaves 131-141.; xiii, 141 leaves illTue, 01 Jan 1974 00:00:00 GMThttp://hdl.handle.net/10125/117511974-01-01T00:00:00ZYamaga, KarenTaxonomy of the marine, luminous bacteriahttp://hdl.handle.net/10125/11750
Abstract: One hundred and seventy-three strains of marine, luminous bacteria isolated from sea water, surfaces and intestines of fish, as well as from the luminous organs of fish and squid were submitted to an extensive phenotypic characterization. A numerical analysis of the results grouped these strains into four clusters which were formed on the basis of overall phenotypic similarity. One cluster, which was given the designation Beneckea harveyi, consisted of strains which had a moles % GC content in their DNAs of 46.5 ± 1.3 and a single, sheathed, polar flagellum when grown in liquid medium. Most of these strains had unsheathed, peritrichous flagella in addition to the sheathed, polar flagellum when grown on solid medium. The two phenotypically similar clusters which were assigned the species designations Photobacterium phosphoreum and f. mandapamensis consisted of strains which had 1-3 unsheathed, polar flagella and moles % GC contents in their DNAs of 41.5 t 0.7 and 42.9 t 0.5, respectively. The cluster designated f. fischeri contained strains having 2-8 sheathed, polar flagella and a moles % GC content of 39.8 t 1.1. These four species could be further distinguished on the basis of a number of nutritional properties as well as other phenotypic traits. The assignment of the luminous, marine bacteria to four species was supported by differences in the properties of the luminous system as well as differences in the pattern of regulation of aspartokinase activity which are discussed. The species B. harveyi was found to be phenotypically similar to a number of previously characterized, non-luminous strains of Beneckea which shou1d probably be assigned to this species.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii at Manoa, 1973.; Bibliography: leaves 78-83.; viii, 83 l illus., tablesMon, 01 Jan 1973 00:00:00 GMThttp://hdl.handle.net/10125/117501973-01-01T00:00:00ZReichelt, John LawrenceOn the microbiology of slime layers formed on immersed materials in a marine environmenthttp://hdl.handle.net/10125/11749
Abstract: A primary film or slime layer forms on immersed surfaces in marine waters; these primary films were assayed for the presence of various microorganisms. In a preliminary study, surface swabbing and filtration techniques were used to identify and quantitate heterotrophic bacterial densities. From these data the succession patterns of representative isolates from seven (tentatively identified) genera common to marine coastal waters were followed. No individual isolate or group dependence upon the chemical composition of the test panel surfaces (which included steel, aluminum, zinc, plexiglass and wood) was found. However, the appearance of various isolates and their relative density were found to vary with the composition of the test material luring the first few days following immersion. Two methods were developed to assay the primary film layers formed on opaque materials immersed in the sea. Each method was designed for expedient field testing and utilized light microscopy of intact, removed, slime layers. In the first method, ultrathin Teflon membranes with micropores were fitted over surfaces of glass, aluminum, phosphor-bronze, zinc, wood and steel. Corrosion products from the metals were shown to diffuse through the membrane pores. Bacteria adsorbed to excised membranes on all surfaces except phosphor-bronze by one day; active proliferation occurred by four days following immersion. Diatoms attached sparingly during the first day but appeared on most all surfaces by four days. Qualitative differences in the bacterial and diatom communities suggested that the surface chemistry may influence microbial attachment. The Parlodion filming technique is the major analytic method used in these studies. It provides a view of the microorganisms exactly as they appear on the test surface. Using this method, quantitative comparisons of bacteria, diatoms and extraneous particulate matter were made for extended periods on aluminum, stainless steel, Monel, glass, plexiglass and phosphor-bronze. Bacteria and diatoms were nonrandomly distributed while extraneous particles showed a (generally) random distribution. Adsorption and attachment sequences consistently began with bacteria, followed by diatoms on all test panel surfaces. Adsorption of bacteria appeared to be dependent upon several factors: the relative polarity and electronegativity of the test material, the ability to synthesize and excrete slimy materials, and the motility or chemotactic response of the attaching bacteria. The evolution of increasingly complex surface ecosystems were compared for all test materials.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii at Manoa, 1972.; Bibliography: leaves 100-105.; ix, 105 l illus., tablesSat, 01 Jan 1972 00:00:00 GMThttp://hdl.handle.net/10125/117491972-01-01T00:00:00ZSechler, Gary EvansThe cytotoxicity of ultraviolet light irradiated reovirushttp://hdl.handle.net/10125/11748
Abstract: When reovirus type 2 was exposed to ultraviolet light (UV) for 2-5 minutes, it acquired a cytotoxic property to HeLa cells. Maximal toxicity was attained after 10 minutes irradiation and decreased to undetectable levels by 60 minutes. The incident photo energy dose at maximum cytotoxicity level was 6 x 10^7 ergs/cm^2. The production of UV-induced cytotoxicity was temperature dependent with a maximum at 37°C. The toxic factor was found to be associated with the virus proteins since it was demonstrated that the "empty" particles of reovirus exposed to ultraviolet light were also toxic to HeLa cells. In addition, studies with virus protein components produced by selective chemical degradation of the mature reovirus with urea showed that the cytotoxicity was associated with the outer capsid proteins. Virions in which the RNA polymerase enzyme was activated by brief heat treatment (70°C, 30 sec) were not toxic to HeLa cells. Extracted RNA exposed to UV-irradiation was also found not to be cytotoxic. Examination of the cytotoxic virus particle indicated no detectable alterations in the following properties: adsorption rates to HeLa cell monolayers, hemagglutination of human "0" erythrocytes, interferon inducing ability, RNA transcriptase activity, virus architecture, buoyant density in Cesium chloride, capsid protein components, viral double-stranded RNA and adenine-rich RNA. In contrast, upon continued irradiation for 60 minutes the loss of cytotoxic property was accompanied by the following: loss of viral hemagglutination, reduction in interferon inducing ability, derangement of the virus architecture, increase in buoyant density, change in electrophoretic mobility of the capsid protein components and alteration in the viral double-stranded RNA. The effect of pre-infecting HeLa cells with viable reovirus before treatment with an equivalent multiplicity of UV-irradiated virus was determined. Viral antigen synthesis and the production of infectious virus was not interfered with to any significant level. Furthermore, the induction of cytotoxicity was not interfered with by pre-infecting the cells with homologous or heterologous viable reovirus. Pre-treatment of mouse L cell cultures with interferon at concentrations which inhibited reovirus yields by 80-90% and vesicular stomatitis virus yields in excess of 99% failed to prevent the reovirus induced cytotoxicity. The toxic property of UV-irradiated reovirus was not dependent on the cell system in which the virus was passed, but was a specific property of the virus itself. All three serotypes of reovirus acquired this toxic property on exposure to ultraviolet light. On examination of the sensitivity of different cell types to the UV-irradiated reovirus induced toxicity, it was found that the established cell lines in general were more sensitive than the primary cell cultures.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1971.; Bibliography: leaves [159]-171.; ix, 171 l illus., tablesFri, 01 Jan 1971 00:00:00 GMThttp://hdl.handle.net/10125/117481971-01-01T00:00:00ZSubasinghe, Don Henry AriyadasaInduction of alpha-glucosidase in Mycoplasma laidlawii Ahttp://hdl.handle.net/10125/11747
Abstract: The object of the study was to find if regulation of gene expression, found so vital to bacteria, exists in the mycoplasma which are noted for their small size, limited biosynthetic ability and small genomes. Glucose grown cells transferred to maltose media exhibit a lag before turbidity or 14C-amino acid incorporation into protein begins to increase. The lag is not observed for glucose grown cells transferred to media containing glucose and maltose or when maltose grown cells are transferred to media containing glucose or maltose. When growth begins the specific activity of a previously unreported alpha-glucosidase increases in the culture transferred to maltose until enzyme levels become 10 fold higher than that measured in the glucose control in which the specific activity was constant. The specific activity stops increasing if chloramphenicol is added. The "differential rate of synthesis" is 10 fold higher in the culture transferred to maltose than in the glucose control. The pH optimum of the alpha-glucosidase is 6080 The specific activity of a previously unreported phosphatase (pH optimum = 6.7, one band in disc electrophoresis at pH 5.5, 7.0, 801, 8.5) is the same and constant in either the glucose or maltose cultures. The presence of glucose, either added with or before maltose, does not interfere with the induction. Partially constitutive mutants were isolated. In one case it was found that a 2 fold increase in basal level of alpha-glucosidase eliminated the lag. Alpha-glucosidase activity paralleled maltose splitting activity (the former being measured by p-nitrophenyl-alpha-glucoside splitting, the latter by enzymatic measurement of glucose released from maltose). The interpretation is that M. laidlwaii A must adapt to maltose metabolism. The adaptation corresponds to the induction of alpha-glucosidase by maltose. The induction is under specific genetic control. There is no "glucose effect" in this system. Maltose metabolism is mediated, at least in part, by alpha-glucosidase in this organism.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1970.; Bibliography: leaves 142-149.; ix, 149 l illus., tablesThu, 01 Jan 1970 00:00:00 GMThttp://hdl.handle.net/10125/117471970-01-01T00:00:00ZSlater, Martin L.Structural studies on chicken IgG immunoglobulinhttp://hdl.handle.net/10125/11746
Abstract: Chicken IgG L chains have a molecular weight of 23,700±800 daltons, and chicken IgG H chains have a molecular weight of 60,800±1400 daltons as determined by high speed sedimentation equilibrium. The sum of the masses of two L chains and two H chains are in excellent agreement with the molecular weight reported for chicken IgG (170,000 daltons). The number of disulfide bonds reduced per molecule of IgG was determined at various concentrations of ME by a titration method. The reduced samples were also analyzed by gel filtration at pH 8.2 and at pH 2.4. A sample of IgG was reduced with 0.01 M ME. About 1.3 disulfide bonds per molecule were reduced. Gel filtration at pH 8.2 showed that no dissociation of the reduced IgG occurred, while by gel filtration at pH 2.4 a relative yield of 4.3% free L chains was obtained. Reduction of chicken IgG with 0.05 to 0.2 MME resulted in the release of free L chains at pH 8.2. Reduction with 0.05 M ME and 0.2 MME reduced 3-4 and 6-7 disulfide bonds per molecule respectively. The gel filtration patterns at pH 2.4 of chicken and human IgG each reduced with 0.01 M ME were similar. Free L chain material was detected in the elution patterns of the two reduced samples. Rabbit IgG was reduced with 0.01 MME and gel filtered at pH 2.4. The elution patterns indicate that most of the IgG were converted to half molecules; no free L chains could be detected. It was concluded that the disulfide bonds between the L and H chains of chicken IgG are more easily reduced than the inter- H chain disulfide bonds. Papain cleaved chicken 19G into Fab and Fc fragments and smaller peptides. The Fab fragment had a molecular weight of 55,300 daltons. The Fc fragment had a molecular weight of 56,900 daltons and was crystallizable. Digestion of chicken 19G with pepsin at pH 4.5 resulted in the conversion to 3.5 S material generally. Small amounts of 5.5 S material (F(ab ' )2) was isolated from pepsin digested chicken 19G euglobulins at pH 4.5 and from 19G pseudoglobulin digested at pH 5.0. The 3.5 S material from five-hour digests contained both Fab' and Fc-like components. After 18 hours of digestion, only 3.5 S Fab' fragments were detected. The yields of various fragments from pepsin digested chicken 19G were dependent upon the digestion time, pH, and type of 19G preparation. Undigested 19G, Fab-like and Fc-like fragments were detected in tryptic digests of chicken 19G at pH 7.2. By ultracentrifugation, 7 S, 5 S, and 3.5 S material were observed. The 5 S material was not isolated. The effect of high salt (1.5 M NaCl) on the aggregation of chicken Fab, Fc, and F(ab')2 was investigated. The Fab and F(ab')2 were not affected by salt concentration with respect to aggregation, while the Fc fragment was completely precipitated in high salt. The relationship on the salt-induced phenomenon to the dependence of high salt of the chicken precipitin reaction was discussed and a hypothetical model for this relationship was presented. Peptide maps of chicken Fab, Fc, H chains, and L chains were prepared. Chicken Hand L chains appear to possess the same variability with regard to heterogeneity as rabbit Hand L chains, respectively. Most of the chicken H chains (60-80%) possessed alanine as the amino-terminal residue. Only 6% of the L chains had alanine at the amino-terminal position. No other amino acids were detected at the amino-terminus.
Description: Typescript.; Vita.; Thesis (Ph. D.)--University of Hawaii, 1970.; Bibliography: leaves [192]-202.; x, 202, [1] l illus., graphs, tablesThu, 01 Jan 1970 00:00:00 GMThttp://hdl.handle.net/10125/117461970-01-01T00:00:00ZKubo, Ralph Teruo, 1942Role of pH in the metabolism of Thiobacillus thiooxidanshttp://hdl.handle.net/10125/11745
Abstract: The pH for the maximal growth of Thiobacillus thiooxidans was found to be 2.0 to 3.0. The cultures failed to grow when the pH was maintained at 1.0 even up to 390 hours. Such cultures resumed growth after changing the pH to 3.0. It was shown that the cells of T. thiooxidans can consume oxygen and oxidize freshly added 35Sofor at least eight hours at pH 1.0. These observations disproved the hypothesis of pH-dependent steps in the initial stages of the sulfur oxidation process. Cultures pre-adjusted to pH 1.0 failed to fix 14CO2, but 14CO2 fixing cultures continued to fix 14CO2 for nearly half an hour at approximately the same rate on changing the pH from 2.0 to 1.0. This suggests that components of the CO2 fixing system 'become unavailable at pH 1.0 since cell-free extracts With added AT.P and Ribose-5-phosphate fix CO2 at pH 1.2. The assimilation of 2-l4C-glycerol ceases when the pH of the culture is changed from 2.0 to 1.0. This suggests that at pH 7.0 energy is not available for synthetic processes. The intracellular ATP pool decreases suddenly as a result of changing pH from 2.0 to 1.0. This implicates .ATP as one of the limiting substrates of the CO2 fixation system at pH 1.0 and perhaps also for glycerol assimilation. The decreased pool size of ATP and continued oxidation of sulfur at pH 1.0 indicates the uncoupling of phosphorylation from sulfur oxidation. This uncoupling is explained in terms of pH-dependent conformational changes in the energy producing sites, probably the membranes. It is concluded that To thiooxidans requires an acidic environment to maintain an integrated energy generating system. Addition of 10^-3 M. pyruvic acid to cultures of T. thiooxidans at pH 2.3 resulted in its rapid intracellular accumulation and in the cessation of sulfur oxidation, C02 fixation and oxygen consumption; at pH 7.0 pyruvate neither inhibited oxygen consumption nor accumulated appreciably intracellularly. Pyruvate did not affect the CO2 fixation in cell-free system at pH 7.0. The above data is explained by the intracellular accumulation of pyruvic acid through passive permeation along with the concomitant decrease in the intracellular pH. Hence, it is concluded that pyruvic acid is an inhibitor for T. thiooxidans not because of special features associated with the intermediary metabolism of the organism but because it renders the intracellular environment acidic. Extracellular acidic pH is necessary to maintain an integrated energy generating system in T. thiooxidans, and the prevailing low pH-values in cultures are responsible for the inhibitory action of pyruvate; the cell is permeable to pyruvic acid as an undissociated molecule.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1970.; Bibliography: leaves [69]-72; 72 l illus., tables, graphsThu, 01 Jan 1970 00:00:00 GMThttp://hdl.handle.net/10125/117451970-01-01T00:00:00ZRao, G. SivajiUltrastructure and physiology of germination in Phytophthora parasiticahttp://hdl.handle.net/10125/11744
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1970.; Bibliography: leaves [190]-196.; x, 196 l illusThu, 01 Jan 1970 00:00:00 GMThttp://hdl.handle.net/10125/117441970-01-01T00:00:00ZHemmes, Don E, 1942Induction of catalase in the athiorhodaceaehttp://hdl.handle.net/10125/11743
Abstract: Catalase induction was investigated in various organisms belonging to the family Athiorhodaceae. Catalase induction could be demonstrated only in Rhodopseudomonas spheroides and related locally-isolated organisms. Both RNA and protein synthesis were found to be necessary for the induced synthesis. Although H2O2, the added inducer, was decomposed in the first 5 min., m-RNA synthesis continued up to 30 min. In Rh. spheroides, maximum induction occurred during the early stationary phase, while in TL-1, TL-4 and Rps. D, the induction was during the logarithmic phase of growth. !h. spheroides alone excreted porphyrins into the medium during the stationary phase. Inhibition of porphyrin synthesis by 8-Hydroxy quinoline (4 x 10^-5M) also inhibited catalase induction. The induced synthesis of catalase has been interpreted to require porphyrin synthesis. Growing TL-4 at constant pH decreased the log phase level of induction. Inducibility under these conditions was maximum at pH 7.3. Growing either Rh~ spheroides or TL-4 at constant pH had no effect on the stationary level of induction. Induction of catalase was observed only in those organisms which can effect an oxygen dependent conversion of the carotenoid spheroidene to spheroidenone. Inhibition of this carotenoid conversion by inhibitors like diphenylamine (3 x 10^-4M) or acridine (5 x 10^-4M) also inhibited the catalase induction. Lower concentration (0.5 to 1.0 x 10^-4M) of the inhibitors had a stimulatory effect. The data is discussed in terms of the involvement of carotenoids in the induction of catalase.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1969.; Bibliography: leaves [84]-90.; vi, 90 l illusWed, 01 Jan 1969 00:00:00 GMThttp://hdl.handle.net/10125/117431969-01-01T00:00:00ZShanmugam, Keelnatham T.Genetic studies on the blue-green alga, Anacystis nidulanshttp://hdl.handle.net/10125/11742
Abstract: Inactivation studies on Anacystis nidulans were performed using UV irradiation and nitrosoguanidine. UV irradiation had no effect when the treated culture was first incubated in the light. When the UV irradiated culture was first incubated in the dark for 9 hours, an exponential survival curve was obtained with slope k=0.23 log S/So ergs•cm. The photoreactivable sector was calculated to be one. A slight shoulder in the survival curve was observed. Extrapolation of the asymptote to the y-axis indicated a target number of 2. The response to nitrosoguanidine was found to be concentration dependant. Mutation experiments were conducted mainly with nitrosoguanidine. A variety of mutant phenotypes was found upon induction with nitrosoguanidine treatment. These were minute colony formers (mcf), filamentous forms (snakes or sna), yellow (yel) and blue (blu) , polymixin B (pmb) and kanamycin (kan) resistance. The frequencies of occurrence were: 3.7x10^-3 (mcf), 1.0x10^-3 (sna) , 1.8x10^-3 (yel), 2.98x10^-3 (blu) and 4.8x10^-4 (pmbr). Auxotrophic mutants were not recovered even after screening approximately 8.0x10^4 surviving colonies with the replica plate method. Cell synchrony was induced by a light-dark regimen. The rates of macromolecular synthesis of the synchronous culture were determined. Protein, RNA, and phospholipid fractions increased exponentially. DNA synthesis was periodic. An abrupt increase in the rate of DNA synthesis was seen at 3 hours prior to the cell division periods. The period of DNA synthesis was 6 to 7 hours. The times of abrupt increases in the rate of DNA synthesis were inferred to be the times of initiation of genome at a fixed point of replication. A genetic map was constructed by taking advantage of cyclic DNA synthesis of synchronously dividing cells. Sequential mutagenesis during the period of DNA synthesis yielded unique temporal patterns for specific marker frequencies. The patterns showed an initial plateau, an abrupt increase, then, a second plateau. Slopes of the abrupt increases were calculated. The times of the abrupt increases were then designated as the times of the replication of the cistrons and, correspondingly, the loci of the markers in time units. The loci of six markers were presented as the genetic map of Anacystis nidulans. These were pmbr , blu, yel, sna, mcf, and kanr in that temporal order.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1969.; Bibliography: leaves [97]-101.; viii, 101 l illusWed, 01 Jan 1969 00:00:00 GMThttp://hdl.handle.net/10125/117421969-01-01T00:00:00ZAsato, YukioStructural and antigenic relationships between avian immunoglobulinshttp://hdl.handle.net/10125/11741
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1968.; Bibliography: leaves [197]-208.; xiii, 208 l illus., tablesMon, 01 Jan 1968 00:00:00 GMThttp://hdl.handle.net/10125/117411968-01-01T00:00:00ZLeslie, Gerrie AllenCell organization and ultrastructure during the culmination of cellular slime moldshttp://hdl.handle.net/10125/11740
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1968.; Bibliography: leaves 190-195.; xiii, 195 l illusMon, 01 Jan 1968 00:00:00 GMThttp://hdl.handle.net/10125/117401968-01-01T00:00:00ZGeorge, Robert P.Studies on the REOvirus type 2 - human amnion cell systemhttp://hdl.handle.net/10125/11739
Abstract: REOvirus type 2 (REO-2), strain D-5, and its interaction with « continuous line of human amnion cells (RA) were investigated. The investigations were carried out to obtain information on the effects of several physicochemical agents on the virion, the early interaction between virus and cell, and some of the consequences stemming from this virus-cell interaction. The virion was found to be resistant to ethyl ether (30% and 50%). It was quite stable at temperatures below 250°C, but at 37°C and 60°C, its infectivity titers were reduced by one-half in 5 days and 30 seconds, respectively. For the first 2-3 minutes, sonication of virus preparations at 20 Kc resulted in increased infectivity titers, but a rapid decrease in titers resulted with prolonged treatment. The virus was stable for at least 7 days over a pH range of 2-9 but was rapidly destroyed at pH 11. UV-irradiation inactivated the virus very rapidly, reducing its infectivity titer by one-half in less than 15 seconds. The rate of adsorption of virus to cell was found to be inversely related to the volume of inoculum and directly related to the temperature. The elution of adsorbed virus from cells was found to depend on the exposure multiplicity and the temperature of incubation. At multiplicities of 10 or more, significant elution was observed; at 1 or less, no significant elution occurred. At 370 C, elution occurred but a stable cell-virus complex was formed within 5 minutes. At lower temperatures, elution occurred but at slower rates. No significant desorption occurred at 40 C. Single cycle growth studies of REO-2 in RA cells revealed an eclipse period of 9 hours and maximal yields of virus obtained in about 36 hours. In comparison, REO-3 in RA cells was found to have an eclipse period of 6 hours and maximal yields obtained in 24-26' hours. Immunofluorescent and cytochemical (acridine orange) examinations of REO-2 infected RA cells revealed the following: (1) the appearance of viral antigen as early as 4 hours post-infection (p.i.), (2) a maximal number of antigen-containing cells at 9 hours p.i., (3) the appearance of orthochromatically green-staining inclusion bodies at 6 hours p.i., and (4) maximal number of cells containing inclusion bodies in 12 hours. Infection of RA cells with either active or UV-inactivated REO-2 at low multiplicities (1-5) resulted in the production of an anti-viral substance which was found to possess many characteristics in common with interferon. In addition, REO-2 was found to be sensitive to the inhibitory effect of this anti-viral agent. When HeLa, RA, or BSC-1 cells were exposed to high multiplicities of UV-inactivated REO-2 preparations, rapid death of cells was observed. This cytotoxic phenomenon was found to have the following characteristics: (1) morphological changes were indistinguishable from that due to viral cytopathic effect, (2) first signs of the effect could be seen at about 3-4 hours p.i. and culminated in 9-10 hours, and (3) the intensity and extent of the effect was dependent on exposure multiplicity. Only UV-inactivated virus preparations produced the effect; neither heat-inactivated nor active virus was observed to produce cytotoxicity. Data obtained strongly implicate the UV-inactivated viral particle as the cytotoxic agent. The significance of the new information obtained and some ramifications in REOvirus research are discussed. In addition, suggestions for further investigations are presented.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1968.; Bibliography: leaves 93-98.; xii, 98 l graphsMon, 01 Jan 1968 00:00:00 GMThttp://hdl.handle.net/10125/117391968-01-01T00:00:00ZOie, Herbert KazutoThe lipids of Rhodomicrobium vannieliihttp://hdl.handle.net/10125/11738
Abstract: Cells of Rhodomicrobium vannielii grown at 29° in a lactate-containing medium were extracted at room temperature with organic solvents. This extractable fraction contains the bulk of simple lipids (1.8% of cell dry weight) and complex lipids (phospholipids, 4.2%, and sulfolipid, 0.01%) coenzyme Q (0.09%) and pigments (carotenoids, 1.2%; and bacteriochlorophyll, 1.9%). The cell-residue contains the bound lipids (2.7%) which are liberated by treatment with alkali. The residue also contains poly-β-hydroxybutyric acid (0.2%), which was extracted with boiling CHCl3. CoQ was. determined to be Q9. Bacteriochlorophyll was found to be type a. Vaccenic acid (C18:1 ) is the major fatty acid component in both simple lipid and phospholipid fraction (ca. 90% in each fraction). The bound lipids contain many fatty acid components, including cyclopropane-, branched-, and alpha(α) and beta-(β)-hydroxy fatty acids. Seven carotenoids were found. Rhodopin (61.3% of total carotenoids by weight) and lycopene (20.5%) are the major constituents; the presence of β-carotene (3.4%) makes R. vannielii unique among the photosynthetic bacteria. The rest of the minor carotenoids are spirilloxanthin (11.2%), anhydro-rhodovibrin (1.8%), rhodovibrin (0.5%), and monodemethylated spirilloxanthin (0.5%). R. vannielii synthesizes mainly the normal spirilloxanthin series and is characterized by a high content of hydroxylated carotenoids. Crude phospholipids were separated from the bacteriochlorophyll by acetone treatment after fractionation on silicic acid columns. They were refractionated by TLC on silica gel-H and 8 components were separated; 7 of these contained phosphorus. The nature and amounts of these components were determined. They are phosphatidic acid (1.83% of total phospholipids by weight), bis-phosphatidic acid (6.75%), phosphatidyl ethanolamine (4.5%), an ornithine ester of phosphatidyl glycerol (46.5%), phosphatidyl glycerol (9.7%), an ornithine amide of an undetermined fatty acid (0.95%), phosphatidyl choline (26.5%), and the ornithine ester of lysophosphatidyl glycerol (3.2%).
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1966.; Bibliography: leaves [85]-96.; 96 l illus., tablesSat, 01 Jan 1966 00:00:00 GMThttp://hdl.handle.net/10125/117381966-01-01T00:00:00ZPark, Chong EelThe nature of genetic transformants of Mycoplasma laidlawiihttp://hdl.handle.net/10125/11737
Abstract: Transformation of a streptomycin-resistant marker was effected in the saprophytic mycoplasmas, at frequencies of 5.4 X 10^-6 or 7.6 X 10^-4 'when challenged in broth or by agar overlay, respectively. However, the expression to streptomycin-resistance was unstable. The instability of expression to streptomycin-resistance is attributed to a stable but non-integrated state of the str^r-allele. Furthermore, it is believed that a state of coexistence of the resident str^s-allele and the exogenomic str^r-allele is responsible for the low levels of resistance. Because of this coexistence, complete expression to streptomycin-resistance (50 μg/ml streptomycin) would not be expected. The level of resistance is believed to be dependent upon the quantity of the str^r-allele product(s) synthesized and accumulated by the transformed cell. Evidences indicate that the str^r-allele is stable and functional, although non-replicative, under non-selective conditions, and that it is stable, functional, and replicative under selective conditions. The addition of ribonucleic acids to the transforming DNA enhanced the biological activity of the DNA. The RNA is believed to inhibit DNAse activity in the transforming culture. Because genomic recombinants cannot be distinguished from the spontaneous mutants, in this system of transformation, genetic analyses of the saprophytic mycoplasmas are severely limited.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1967.; Bibliography: leaves [121]-123.; ix, 123 l graphs, tablesSun, 01 Jan 1967 00:00:00 GMThttp://hdl.handle.net/10125/117371967-01-01T00:00:00ZIha, Thomas HirohideClinical and laboratory studies of the epidemiology, etiology, and treatment of urethritis in the malehttp://hdl.handle.net/10125/11736
Abstract: Gonorrhea is one of the most common bacterial diseases of man. In 1966, an estimated 1,500,000 people in the United States acquired gonorrhea. During periods of military conflict, this disease becomes extraordinarily common in military personnel. For example, over 66,000 cases of gonococcal urethritis occurred among U. S. Forces in Viet Nam in 1966. In some areas, the annual incidence of gonorrhea approached 500 cases per 1,000 men. The second most common disease of military personnel is nongonococcal urethritis, a general classification of those urethritides which are not associated with demonstrable N. gonorrhea, but may not have a single etiology. Taken together, gonococcal and nongonococcal urethritis are several times as common as any other reportable infectious disease in military populations in the Far East. The series of studies from which this dissertation is drawn began early in 1965, and is still continuing. The studies were generated by the alarming increase in incidence of urethritis accompanying the Viet Nam build-up; by the unexpectedly high failure rate accompanying standard penicillin therapy of gonorrhea; by the lack of a consistent approach by military physicians to the diagnosis and treatment of nongonococcal urethritis; and by the immediate need for a practical solution to these problems. This research was approved and undertaken for the expressed purpose of finding an adequate treatment for urethritis; and of effecting preventive measures to reduce the incidence of urethritis in military personnel. However, the philosophy of the investigators was that these studies also afforded an opportunity to learn more about the epidemiology, etiology, and pathogenesis of the various urethritides. As the investigation progressed, a "story" became apparent which tied together gonorrhea, postgonococcal urethritis, and nongonococcal urethritis as sequential stages in the development of chronic urethritis in certain situations. It seemed appropriate, therefore, to describe this "story" in a series of manuscripts, dealing with certain aspects of the overall problem of urethritis, but presented in an order which corresponds to the natural sequence with which chronic urethritis often develops in the male. These manuscripts are included as chapters in this dissertation, in the same order in which they were prepared for publication. Chapter three is not yet finished. It is partly based upon recently completed work with gonorrhea infection in women, but is inserted into the dissertation since it pertains to the epidemiology of gonococcal infection in men.
Description: Typescript.; Thesis (Ph. D.)--University of Hawaii, 1967.; Includes bibliographies.; ix, 111 l graphs, tablesSun, 01 Jan 1967 00:00:00 GMThttp://hdl.handle.net/10125/117361967-01-01T00:00:00ZHolmes, King Kennard